Gene expression signature for il-6/stat3 signaling pathway and use thereof

ABSTRACT

The present invention relates to a set of biomarkers, microarrays that provide for detection thereof, an expression signature comprising 16 genes or a subset thereof, and the use thereof in determining the regulation status of IL-6/STAT3 signaling pathway in a cell sample or subject, as well as compositions for the detection thereof. The regulation status of IL-6/STAT3 signaling pathway in a cell sample or subject may be assayed based on the level of expression of one or more of these genes. The methods and compositions provided herein may be used to evaluate IL-6/STAT3 pathway regulation status in a sample; classify a cell sample as having a deregulated or regulated IL-6/STAT3 signaling pathway; determine whether an agent modulates the IL-6/STAT3 signaling pathway; predict the response of a subject to an agent that modulates the IL-6/STAT3 signaling pathway; assign treatment to a subject; and/or evaluate the pharmacodynamic effects of therapies designed to regulate IL-6/STAT3 pathway signaling. Expression of the biomarkers is preferably determined by RT-PCR using SYBR Green methods, and the expression data analyzed and compared to a control sample by use of the random forest method.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to provisional application Ser. No.61,642,037, filed on May 3, 2012, the disclosure of which, including allsequence information, is incorporated by reference herein.

BACKGROUND

1. Field of the Invention

The present invention relates to a novel set of markers, microarrayscontaining these markers, and an expression signature comprising 16genes or a subset thereof and the use thereof in determining theregulation status of IL-6/STAT3 signaling pathway in a cell sample orsubject, as well as compositions for the detection thereof. Theregulation status of IL-6/STAT3 signaling pathway in a cell sample orsubject may be assayed based on the level of expression of one or moreof these genes. More specifically, the invention provides a set of geneswhich can be used as biomarkers and as gene signatures for evaluatingIL-6/STAT3 pathway regulation or deregulation in a sample; diagnosticand/of classification of a sample, e.g., tumor, as having a deregulatedIL-6/STAT3 signaling pathway; determining whether an agent modulates theIL-6/STAT3 signaling pathway in a sample; predicting the response of asubject to an agent that modulates the IL-6/STAT3 signaling pathway;assigning treatment to a subject; and evaluating the pharmacodynamiceffects of therapies designed to target the IL-6/STAT3 pathway. The geneexpression signature may be used with companion algorithms to provide aquantitative measure of IL-6/STAT3 pathway activity. Expression of theprovided biomarkers is preferably determined by RT-PCR using SYBR green,and the expression data analyzed and compared to a control sample by useof the Random Forest method.

2. Description of Related Art

The STAT (Signal Transducer and Activator of Transcription) familyconsists of seven mammalian members. Originally, STAT proteins wereidentified as intracellular signaling mediators of cytokine signals.Every STAT family member responds to a defined set of cytokines.Interestingly, STAT3 is known to be activated by IL-6 (Yu H, Pardoll D,Jove R. STATs in cancer inflammation and immunity: a leading role forSTAT3. Nat Rev Cancer. 2009 November; 9(11):798-809).

STAT proteins are latent cytoplasmic transcription factors that requirephosphorylation for nuclear retention. Engagement of IL-6 to itsspecific receptor IL-6R (IL-6 receptor) activates receptor-associatedtyrosine kinase, such as Janus Kinase 2 (JAK2). Activated JAK2 in turnphosphorylates tyrosine residues in the cytoplasmic tail of the IL-6receptor that function as docking sites for STAT3. JAK2 dependentphosphorylation of STAT3 leads to its homodimerization and nucleartranslocation, where activated STAT3 function as transcriptionalactivator, inducing expression of target genes (Levy D E, Darnell J EJr. Stats: transcriptional control and biological impact. Nat Rev MolCell Biol. 2002 September; 3(9):651-62).

IL-6/STAT3 has been implicated as crucial mediator for inflammatoryresponse (Grivennikov S I, Karin M. Dangerous liaisons: STAT3 andNF-kappaB collaboration and crosstalk in cancer. Cytokine Growth FactorRev. 2010 February; 21(1):11-9. Epub 2009 Dec. 16). Moreover,deregulated IL-6/STAT3 signaling has been associated with biologicalevents such as embryonic development, programmed cell death,organogenesis, innate immunity, adaptive immunity and cell growthregulation in many organisms (Mankan A K, Greten F R. Inhibiting signaltransducer and activator of transcription 3: rationality and rationaledesign of inhibitors. Expert Opin Investig Drugs. 2011 September;20(9):1263-75. Epub 2011 Jul. 14). In addition, STAT3 plays an essentialrole in cancer initiation and progression by selectively inducing andmaintaining a pro-carcinogenic inflammatory microenvironment (Yu H,Pardoll D, Jove R. STATs in cancer inflammation and immunity: a leadingrole for STAT3. Nat Rev Cancer. 2009 November; 9(11):798-809).

Perturbation of the IL-6/STAT3 signaling pathway causes a change inSTAT3 transcriptional activity and, in turn, alters the expression levelof STAT3 target genes. Although changes in gene expression of STAT3target genes can serve as indicators of IL-6/STAT3 pathway activity,real time PCR assay based methods are not yet available toquantitatively measure IL-6/STAT3 pathway activity.

The identification of patient subpopulations most likely to respond totherapy is a central goal of modem molecular medicine. This notion isparticularly important for cancer due to the large number of approvedand experimental therapies (Rothenberg et al., 2003, Nat. Rev. Cancer3:303-309), low response rates to many current treatments, and clinicalimportance of using the optimal therapy in the first treatment cycle(Dracopoli, 2005, Curr. Mol. Med. 5:103-110). In addition, the narrowtherapeutic index and severe toxicity profiles associated with currentlymarketed cytotoxics results in a pressing need for accurate responseprediction. Although recent studies have identified gene expressionsignatures associated with response to cytotoxic chemotherapies(Folgueria et al., 2005, Clin. Cancer Res. 11:7434-7443; Ayers et al.,2004, 22:2284-2293; Chang et al., 2003, Lancet 362:362-369; Rouzier etal., 2005, Proc. Natl. Acad. Sci. USA 102: 8315-8320), these examples(and others from the literature) remain unvalidated and have not yet hada major effect on clinical practice. In addition to technical issues,such as lack of a standard technology platform and difficultiessurrounding the collection of clinical samples, the myriad of cellularprocesses affected by cytotoxic chemotherapies may hinder theidentification of practical and robust gene expression predictors ofresponse to these agents. One exception may be the recent finding bymicroarray that low mRNA expression of the microtubule-associate proteinTau is predictive of improved response to paclitaxel (Rouzier et al.,supra).

To improve on the limitations of cytotoxic chemotherapies, currentapproaches to dnig design in oncology are aimed at modulating specificcell signaling pathways important for tumor growth and survival (Hahnand Weinberg, 2002, Nat. Rev. Cancer 2:331-341; Hanahan and Weinberg,2000, Cell 100:57-70; Trosko et al., 2004, Ann. N.Y. Acad. Sci.1028:192-201). In cancer cells, these pathways become deregulatedresulting in aberrant signaling, inhibition of apoptosis, increasedmetastasis, and increased cell proliferation (reviewed in Adjei andHildalgo, 2005, J. Clin. Oncol. 23:5386-5403). Although normal cellsintegrate multiple signaling pathways for controlled growth andproliferation, tumors seem to be heavily reliant on activation of one ortwo pathways (“oncogene activation”). The components of these aberrantsignaling pathways represent attractive selective targets for newanticancer therapies. In addition, responder identification for targettherapies may be more achievable than for cytotoxics, as it seemslogical that patients with tumors that are “driven” by a particularpathway will respond to therapeutics targeting components of thatpathway. Therefore, it is crucial that methods to identify the pathwaysthat are active in particular tumors are developed, and this informationused to guide therapeutic decisions. Identification of gene expressionprofiles that are indicative of pathway activation status is one way toachieve this goal.

Given its involvement in numerous biological functions and diseases, agene expression signature-based readout of IL-6/STAT3 pathway activationmay be more appropriate than relying on a single indicator of pathwayactivity, as the same signature of gene expression may be elicited byactivation of multiple components of the pathway.

Based on the foregoing, a reliable method for accurately andquantitatively assessing the IL-6/STAT3 pathway activation status in abiological sample or individual would be beneficial given the apparentrole of this pathway in different disease conditions. Particularly,given its involvement in numerous biological functions and diseases, agene expression signature-based readout of IL-6/STAT3 pathway activationmay be more appropriate and predictive than relying on a singleindicator of pathway activity, as the same signature of gene expressionmay be elicited by activation of multiple components of the pathway.

SUMMARY

Signaling pathways play central roles in cellular physiology, andassessing the state of these pathways can help to clarify the molecularmechanisms of disease, non-cancer inflammatory conditions, and theinflammatory response. However, a multitude of components can activate,modify, and/or inhibit IL-6/STAT3 signaling at multiple points along thepathway and/or may be involved in crosstalk with other pathways. As aresult, measuring pathway activity using traditional methods that onlytest a few well-characterized pathway components may miss otherimportant pathway mediators. Conversely, multi-gene expression basedmethods measure pathway alteration as a function of the downstreameffect of pathway regulation on multiple gene expression changes, thusenabling reliable measurement of pathway activity. These downstream geneexpression alterations can potentially capture all changes related toany upstream alteration of a pathway component.

The present invention satisfies these unmet needs and describes sets ofgenes that provide a gene signature for evaluating Notch pathwayactivity. These gene sets were identified from an initial set of 88genes derived from microarray profiling on human liver hepatocellularcarcinoma cells (HepG2) and human mammary epithelial cells (MCF10A)treated with IL-6 and siRNA targeting STAT3 (FIG. 2A-B). Genespotentially to be included in the Notch gene signature were selected onthe basis of statistically significant expression changes in response toIL-6 and reversion of altered expression upon treatment with STAT3siRNA. These 88 response genes were further verified by SYBR Green basedreal-time PCR using a 16 sample training set in which nine sample werestimulated with IL-6 to activate pathway activity and seven samples weretreated with STAT3 siRNA to reverse the activation of pathway activityby IL-6 (FIG. 3A-B). Using a mathematical classifier method, preferablyusing Random Forest method, a panel of 16 genes was identified as geneexpression signatures for assessing the regulatory status of theIL-6/STAT3 pathway activity. The utility of the 16 gene signature wasverified on these samples by cross validation with a Random Forestmethod and 87.5% of the samples were classified correctly (FIG. 5A-B).

The invention provides compositions for detection of the regulationstatus of IL-6/STAT3 signaling pathway in a cell sample or subject,comprising primers that amplify at least 5 of the genes selected fromthe group consisting of STAT3, SOCS3, IFITM2, CEBPD, JUNB, TUBB2A,IL-6ST, CASP4, PROS1, TNFRSF1A, PVRL2, PHF21A, BCL3, NRP1, GLRX, andTGM2 or an ortholog or variant thereof.

In one embodiment, the primers amplify at least 5, at least 6, at least7, at least 8, at least 9, at least 10, at least 11, at least 12, atleast 13, at least 14, or at least 15 of said genes. Preferably, thecomposition includes primers for amplification of at least 10, at least11, at least 12, at least 13, at least 14, or at least 15 of said genes.For example, the composition may include primers for amplification of atleast 10 of said genes. Most preferably, the composition includesprimers for amplification of all 16 of said genes.

In another embodiment, the primers are in contact with the sample to betested for the level of IL-6/STAT3 pathway activity. In one embodiment,at one of said primers comprises a fluorophore and matched fluorescencequencher. The primers may be contained in one or more wells of amulti-well reaction vessel. Additionally, the primers for amplificationof at least two of said genes can be included together in a duplex ormultiplex reaction.

In one embodiment, the primers include at least five primer pairsselected from the group consisting of:

TGACATGGAGTTGACCTCG and CTGGAACCACAAAGTTAGTAGTTTC; CCACCTACTGAACCCTCCTCCand TCTTCCGACAGAGATGCTGAA; TCCCACGTACTCTATCTTCCATTC andCTGATGCAGGACTCGGCTG; CGCCATGTACGACGACGAG and CGCCTTGTGATTGCTGTTG;CGACTACAAACTCCTGAAACCG and GAAGAGGCGAGCTTGAGAGAC; AACTTCTCAGATCAATCGTGCand AGACCATGCTTGAGGACAAC; AAGATTTGAAACAGTTGGCATGGAG andCCTTCACTGAGGCATGTAGC; GAGAGACAGCACAATGGGCTC andCTTCCGAAATACTTCCTCTAGGTG; ATCGGATACAGGCCCTAAGTC andTTGTCCAAGACGGCAAGTTG; TGTTACACTAATAGAAACTTGGCAC andCCTTAGGACAGTTCAGCTTGC; AAGCCAAAGAGACTCAGGTG and CAGGTATCAGGGCTGGTTCCTC;GGCAGAAGGAGATGCACAGC and TCAGAGTCTACAGGTTTGGAGAG;CACTCTCTACCAGATAACTGAGGAG and TAATAATTTACATCGTGATCCGTGC;CAACAACTATGATACACCTGAGC and TTCCACTTCACAGCCCAGC; GCAGAGGCTGTGGTCATGC andTGCTTTAATCTTTGCTGGTAGTC; and CTTCACAAGGGCGAACCAC andGCGGCAGACGTACTCCTCAG.

In another embodiment, the amplified genes are at least 90% identical toor specifically hybridize to at least 5 genes having accession numbersselected from the group consisting of NM_(—)213662, NM_(—)003955,NM_(—)006435, NM_(—)005195, NM_(—)002229, NM_(—)001069, NM_(—)002184,NM_(—)001225, NM_(—)000313, NM_(—)001065, NM_(—)002856, NM_(—)016621,NM_(—)005178, NM_(—)003873, NM_(—)002064, and NM_(—)004613. Preferably,the amplified genes are at least 5 genes having accession numbersselected from the group consisting of NM_(—)213662, NM_(—)003955,NM_(—)006435, NM_(—)005195, NM_(—)002229, NM_(—)001069, NM_(—)002184,NM_(—)001225, NM_(—)000313, NM_(—)001065, NM_(—)002856, NM_(—)016621,NM_(—)005178, NM_(—)003873, NM_(—)002064, and NM_(—)004613.

The compositions of the invention can further comprise primers fordetecting the expression level of between 1 and 10 housekeeping genes(e.g., 5 housekeeping genes).

Additionally, the composition can further comprise a DNA or RNApolymerase. In one embodiment, these compositions are adapted foreffecting PCR, real-time PCR, strand displacement amplification (SDA),loop-mediated isothermal amplification (LAMP), rolling circleamplification (RCA), transcription-mediated amplification (TMA),self-sustained sequence replication (3SR), nucleic acid sequence basedamplification (NASBA), reverse transcriptase polymerase chain reaction(RT-PCR), or helicase-dependent isothermal DNA amplification.

In another embodiment, the compositions further comprise a double strandnucleic acid-specific dye that is used for detecting the level ofexpression. Exemplary double strand nucleic acid specific dyes include,but are not limited to, SYBR Green I, SYBR Gold, ethidium bromide,propidium bromide, Pico Green, Hoechst 33258, YO-PRO-I and YO-YO-I,Boxto, Evagreen, LC Green, LC Green Plus, and Syto 9. Preferably, thecomposition is effected for real-time PCR amplification with detectionby a SYBR Green method.

Moreover, the invention encompasses methods for the detection ofIL-6/STAT3 signaling pathway activity or regulation status in a cellsample or subject, comprising use of the inventive compositions. Suchmethods comprise using these compositions to amplify and detect thelevel of expression of at least five genes selected from the groupconsisting of STAT3, SOCS3, IFITM2, CEBPD, JUNB, TUBB2A, IL-6ST, CASP4,PROS1, TNFRSF1A, PVRL2, PHF21A, BCL3, NRP1, GLRX, and TGM2 or anortholog or variant thereof. Detecting the level of expression can beeffected by a method comprising amplification of mRNA of said at leasttwo genes. Preferably, such detection is accomplished using a doublestrand nucleic acid-specific dye. Non-limiting, exemplary double strandnucleic acid specific dyes include SYBR Green I, SYBR Gold, ethidiumbromide, propidium bromide, Pico Green, Hoechst 33258, YO-PRO-I andYO-YO-I, Boxto, Evagreen, LC Green, LC Green Plus, and Syto 9.Amplification can be effected by a method comprising PCR, real-time PCR,strand displacement amplification (SDA), loop-mediated isothermalamplification (LAMP), rolling circle amplification (RCA),transcription-mediated amplification (TMA), self-sustained sequencereplication (3SR), nucleic acid sequence based amplification (NASBA),reverse transcriptase polymerase chain reaction (RT-PCR), orhelicase-dependent isothermal DNA amplification.

Preferably, the methods further comprise comparing the level ofexpression of said genes in the sample or subject to the level ofexpression of said genes in a control sample. In one embodiment, thelevels of expression are classified using a mathematical classifiermethod to determine the regulation status of IL-6/STAT3 signalingpathway in the in the sample or subject as compared to the controlsample. Preferably, the mathematical classifier is a Random Forestmethod.

As discussed above, the compositions employed in the methods can includeprimers that comprise a fluorophore and matched fluorescence quencher.Additionally, such primers may be contained in one or more wells of amulti-well reaction vessel. In one embodiment, the primers foramplification of at least two of said genes are included together in aduplex or multiplex reaction. The methods may further comprise detectingthe expression level of between 1 and 10 housekeeping genes.

The invention also encompasses methods for determining the level ofactivity or regulation status of the IL-6/STAT3 signaling pathway in acell sample or subject by (1) detecting the expression level of at least5 of the genes selected from the group consisting of STAT3, SOCS3,IFITM2, CEBPD, JUNB, TUBB2A, IL-6ST, CASP4, PROS1, TNFRSFIA, PVRL2,PHF21A, BCL3, NRP1, GLRX, and TGM2 or an ortholog or variant thereof ina cell sample or subject, e.g., using SYBR Green based real-time PCR.The expression level of the genes in the cell sample or subject can becompared to the expression level of the same genes in a control sample,such that the level of activity or regulation status of the IL-6/STAT3signaling pathway in a cell sample or in a subject is determined basedon this comparison. In one aspect, the ortholog or variant possesses atleast 80, 85, 90, or 95% sequence identity to one of the recited genes.In another aspect, the ortholog is a rodent or non-human primate gene.

In particular, methods of determining the level of activity orregulation status of IL-6/STAT3 signaling pathway in a cell sample or ina subject are provided. The methods comprise: (a) detecting theexpression of at least 2 genes selected from the group consisting ofSTAT3, SOCS3, IFITM2, CEBPD, JUNB, TUBB2A, IL-6ST, CASP4, PROS1,TNFRSFIA, PVRL2, PHF21A, BCL3, NRP1, GLRX, and TGM2, or an ortholog orvariant thereof, in a cell sample or subject; and (b) comparing theexpression level of the genes in the cell sample or subject to theexpression level of the same genes in a control cell sample, wherein thelevel of activity or regulation status of the IL-6/STAT3 signalingpathway in a cell sample or subject is determined based on thiscomparison. Preferably, at least two primer pairs selected from thegroup consisting of:

TGACATGGAGTTGACCTCG and CTGGAACCACAAAGTTAGTAGTTTC; CCACCTACTGAACCCTCCTCCand TCTTCCGACAGAGATGCTGAA; TCCCACGTACTCTATCTTCCATTC andCTGATGCAGGACTCGGCTG; CGCCATGTACGACGACGAG and CGCCTTGTGATTGCTGTTG;CGACTACAAACTCCTGAAACCG and GAAGAGGCGAGCTTGAGAGAC; AACTTCTCAGATCAATCGTGCand AGACCATGCTTGAGGACAAC; AAGATTTGAAACAGTTGGCATGGAG andCCTTCACTGAGGCATGTAGC; GAGAGACAGCACAATGGGCTC andCTTCCGAAATACTTCCTCTAGGTG; ATCGGATACAGGCCCTAAGTC andTTGTCCAAGACGGCAAGTTG; TGTTACACTAATAGAAACTTGGCAC andCCTTAGGACAGTTCAGCTTGC; AAGCCAAAGAGACTCAGGTG and CAGGTATCAGGGCTGGTTCCTC;GGCAGAAGGAGATGCACAGC and TCAGAGTCTACAGGTTTGGAGAG;CACTCTCTACCAGATAACTGAGGAG and TAATAATTTACATCGTGATCCGTGC;CAACAACTATGATACACCTGAGC and TTCCACTTCACAGCCCAGC; GCAGAGGCTGTGGTCATGC andTGCTTTAATCTTTGCTGGTAGTC; and CTTCACAAGGGCGAACCAC andGCGGCAGACGTACTCCTCAGare used to detect the expression of the at least 2 genes.

In one embodiment, gene expression is assayed by real timeamplification, which preferably comprises SYBR Green based real-timePCR. The resulting gene expression data is preferably analyzed using aΔΔCt method and, optionally, further analyzed using a Random Forestmethod.

In one embodiment, a cell sample is obtained from a patient or non-humananimal that is potentially to be treated with a compound or therapy thatmodulates IL-6/STAT3 signaling and the method is used to predict whethersaid patient or non-human animal will respond to treatment with saidcompound or therapy. In another embodiment, a cell sample is obtainedfrom a patient or non-human animal that has been treated with a compoundor therapy that modulates IL-6/STAT3 signaling and the method is used toassess the efficacy of the treatment protocol.

These methods may be used to evaluate the regulatory status ofIL-6/STAT3 pathway in a sample; classify a cell sample as having aderegulated or regulated IL-6/STAT3 signaling pathway; determine whetheran agent modulates the IL-6/STAT3 signaling pathway in sample; predictthe response of a subject to an agent that modulates the IL-6/STAT3signaling pathway (which can be used to assign treatment to a subject);evaluate the pharmacodynamic effects of therapies designed to regulateIL-6/STAT3 pathway signaling; evaluate the pharmacodynamic effects oftherapies for treatment of a disease associated with IL-6/STAT3 pathwaydysregulation; evaluate toxicity of an agent a compound or therapy thatmodulates IL-6/STAT3 signaling; detect a disease associated withIL-6/STAT3 pathway dysregulation; identify a disease associated withIL-6/STAT3 pathway dysregulation and/or diagnose a disease associatedwith IL-6/STAT3 pathway dysregulation or a subject at risk of developinga disease associated with IL-6/STAT3 pathway dysregulation (which can beused to treat said patient for said disease); assign treatment to asubject having a disease associated with IL-6/STAT3 pathwaydysregulation; predict treatment outcome for a subject having a diseaseassociated with IL-6/STAT3 pathway dysregulation; monitor treatmentefficacy in a subject having a disease associated with IL-6/STAT3pathway dysregulation; and/or detect inflammation sites in vivo or exvivo.

In one embodiment, the methods are used to assess a pre-malignant orcancerous inflammatory condition or other disease involving aberrantcell proliferation characterized by IL-6/STAT3 pathway dysregulation(e.g., a precancerous condition, cancer or metastases). Non-limitingexamples of cancer include lung, breast, esophageal, head and neck,colonic, gastrointestinal, prostate, multiple myeloma, hepatic, ovarian,neuroblastoma, glioblastoma, melanoma, pancreatic adenocarcinoma, renalcell carcinoma, cholangiocellular carcinoma, and various leukemias andlymphomas. In another embodiment, the methods are used to identify anon-cancer inflammatory condition or disease characterized by IL-6/STAT3pathway dysregulation. Non-limiting examples of non-cancer inflammatoryconditions include hypoferremia of inflammation, acute-phase response toinflammation and infection, chronic inflammation, inflammation incardiovascular, systemic juvenile rheumatoid arthritis, Staphylococcusepidermidis-induced peritoneal inflammation, and pulmonary inflammation(e.g., adult respiratory distress syndrome, shock lung, chronicpulmonary inflammatory disease, pulmonary sarcoidosis, pulmonaryfibrosis and silicosis).

Additionally, these methods can be used in a screen for compounds whichmodulate Notch signaling pathway activity. In one embodiment, suchscreening methods comprise contacting one or more cells with a compoundthat potentially modulates Notch pathway activity and detecting thelevel of activity or regulation status of the Notch signaling pathway insaid cells, and, based thereon, identifying said compound as a compoundthat modulates Notch pathway activity. Preferably, one or more cells arefurther contacted with an agent known to affect Notch pathway activity.

The invention also encompasses one or more gene expression data setsobtained using the inventive methods. These gene expression data setscan be derived from the same individual or from different individuals.In particular, the expression data sets can be derived from the same ordifferent individual treated with a particular agent or therapeuticregimen. Preferably, the gene expression data sets are annotated toidentify one or more variables such as gender, age, disease condition,HLA type, treatment regimen, genetic deficiency. In one embodiment, thegene expression data set is suitable for use as part of a therapeuticassessment and/or design of a treatment regimen and/or the design of atherapeutic planning regimen. Moreover, the invention encompassesmethods of using such gene expression set as part of a therapeuticassessment and/or design of a treatment regimen.

DETAILED DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic depicting an overview of the experiments thatresulted in identification of a unique gene signature profile of theIL-6/STAT3 pathway.

FIG. 2 depicts the result of experiments wherein HepG2 and MCF10A cellswere transfected with siRNA targeting STAT3 for 72 hours and IL-6 wasadded in the last 8 hours. Activation of IL-6/STAT3 pathway wasconfirmed in both cell lines by increased mRNA expression levels ofIL-6/STAT3 targeting genes (Panel B). The effect of STAT3 targetingsiRNA was verified by decreased mRNA expression levels of STAT3 in HepG2and MCF10A cells (Panel A).

FIG. 3 depicts whole genome microarray analysis used to identify theeighty-eight (88) IL-6/STAT3 response genes. Panels A and B show a listof IL-6/STAT3 response genes identified using HepG2 cells, while Panel Cshows a list of IL-6/STAT3 response genes identified using MCF10A cells.

FIG. 4 depicts protein expression levels in sixteen (16) samples, whichwere used as a training dataset for IL-6/STAT3 pathway gene signatureidentification. Nine samples were stimulated with IL-6 (Panel A), andwestern blot analysis confirmed increased STAT3 protein levels. Sevensamples were transfected with siRNA targeting STAT3 (Panel B), andwestern blot analysis confirmed decreased STAT3 protein levels in bothphosphorylated and total forms.

FIG. 5 contains results of cross validation of the gene expressionsignature of the IL-6/STAT3 pathway. A heat-map depicts the PCRexpression data of the 16 signature genes across sixteen samples (PanelA). The gene signature was verified by cross validation on 16 sampleswith Random Forest Machine classification method (Panel B). The genesignature correctly predicted the regulatory status of fourteen out ofsixteen samples in the cross-validation process (Panel B; blue,positively regulated samples; red, negatively regulated samples; gray,untreated samples).

FIG. 6 contains the sequence information for all primers used tovalidate the 88 TL-6/STAT3 response genes using real-time PCR.

DETAILED DESCRIPTION

Prior to disclosing the invention in detail the following definitionsare provided. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood to one ofordinary skill in the art to which this invention belongs.

As used herein, oligonucleotide sequences that are complementary to oneor more of the genes described herein, refers to oligonucleotides thatare capable of hybridizing under stringent conditions to at least partof the nucleotide sequence of said genes. Such hybridizableoligonucleotides will typically exhibit at least about 75% sequenceidentity at the nucleotide level to said genes, preferably about 80% or85% sequence identity, or more preferably about 90% or 95% or moresequence identity to said genes.

“Bind(s) substantially” refers to complementary hybridization between aprobe nucleic acid and a target nucleic acid and embraces minormismatches that can be accommodated by reducing the stringency of thehybridization media to achieve the desired detection of the targetpolynucleotide sequence.

The phrase “hybridizing specifically to” refers to the binding,duplexing or hybridizing of a molecule substantially to or only to aparticular nucleotide sequence or sequences under stringent conditionswhen that sequence is present in a complex mixture (e.g., totalcellular) DNA or RNA.

“Biomarker” means any gene, protein, or an EST derived from that gene,the expression or level of which changes between certain conditions.Where the expression of the gene correlates with a certain condition,the gene is a biomarker for that condition.

“Biomarker-derived polynucleotides” means the RNA transcribed from abiomarker gene, any cDNA or cRNA produced therefrom, and any nucleicacid derived therefrom, such as synthetic nucleic acid having a sequencederived from the gene corresponding to the biomarker gene.

“Primer” refers to a polynucleotide or polynucleotide analog having asequence which base-pairs to a second polynucleotide and can be used toprime synthesis of the complement thereof, e.g., synthesis by a reversetranscriptase, thermostable DNA polymerase, or other DNA or RNApolymerase. Frequently primers are used in pairs, i.e., a forwards andreverse primer which base pair with the opposite ends (and on thecomplementary strands) of a sequence to be amplified. The length of aprimer may vary, and typically includes a region of sufficient length toconfer specific base pairing under the applicable reaction conditions.For example, a typical primer for use in an RT-PCR may have acomplementary sequence of a length between 19 and 25 bases, though theprimer length may be longer or shorter depending upon the cyclingtemperatures, Tm, CG content, complexity of the nucleic acids in thesample, etc. A primer may also optionally include a non-complementarysequence (most typically at the 5′ end), for example, to produce aproduct containing said non-complementary sequence. A primer may alsoinclude one or more modifications, such as the addition of a fluorophoreand/or a matched quencher (including the fluorophore and quencher pairsshown in Table 1 herein). Primers typically comprise a DNA sequence butmay include other nucleic acids or nucleic acid analogs, e.g., RNA,peptide-nucleic acids (PNAs), chimeric molecules comprising one or moreDNA, RNA, and/or PNA bases, etc. A PNA oligonucleotide refers to anoligonucleotide wherein the sugar-backbone is substituted with an amidecontaining backbone, in particular an aminoethylglycine backbone. Thebases are retained and are bound directly or indirectly to aza nitrogenatoms of the amide portion of the backbone (see U.S. Pat. Nos.5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference). Other modifications which may be included ina primer are disclosed in U.S. Pat. No. 6,303,374.

A gene marker is “informative” for a condition, phenotype, genotype orclinical characteristic if the expression of the gene marker iscorrelated or anti-correlated with the condition, phenotype, genotype orclinical characteristic to a greater degree than would be expected bychance.

As used herein, the term “gene” has its meaning as understood in theart. However, it will be appreciated by those of ordinary skill in theart that the term “gene” may include gene regulatory sequences (e.g.,promoters, enhancers, etc.) and/or intron sequences. It will further beappreciated that definitions of gene include references to nucleic acidsthat do not encode proteins but rather encode functional RNA moleculessuch as tRNAs. For clarity, the term gene generally refers to a portionof a nucleic acid that encodes a protein; the term may optionallyencompass regulatory sequences. This definition is not intended toexclude application of the term “gene” to non-protein coding expressionunits but rather to clarify that, in most cases, the term as used inthis document refers to a protein coding nucleic acid. In some cases,the gene includes regulatory sequences involved in transcription, ormessage production or composition. In other embodiments, the genecomprises transcribed sequences that encode for a protein, polypeptideor peptide. In keeping with the terminology described herein, an“isolated gene” may comprise transcribed nucleic acid(s), regulatorysequences, coding sequences, or the like, isolated substantially awayfrom other such sequences, such as other naturally occurring genes,regulatory sequences, polypeptide or peptide encoding sequences, etc. Inthis respect, the term “gene” is used for simplicity to refer to anucleic acid comprising a nucleotide sequence that is transcribed, andthe complement thereof. In particular embodiments, the transcribednucleotide sequence comprises at least one functional protein,polypeptide and/or peptide encoding unit. As will be understood by thosein the art, this functional term “gene” includes both genomic sequences,RNA or cDNA sequences, or smaller engineered nucleic acid segments,including nucleic acid segments of a non-transcribed part of a gene,including but not limited to the non-transcribed promoter or enhancerregions of a gene. Smaller engineered gene nucleic acid segments mayexpress, or may be adapted to express using nucleic acid manipulationtechnology, proteins, polypeptides, domains, peptides, fusion proteins,mutants and/or such like. The sequences which are located 5′ of thecoding region and which are present on the mRNA are referred to as 5′untranslated sequences (“5′UTR”). The sequences which are located 3′ ordownstream of the coding region and which are present on the mRNA arereferred to as 3′ untranslated sequences, or (“3′UTR”).

“Signature” refers to the differential expression pattern. It could beexpressed as the number of individual unique probes whose expression isdetected when a cRNA product is used in microarray analysis. It couldalso be expressed as the number of individual genes whose expression isdetected with real time RT-PCR. A signature may be exemplified by aparticular set of biomarkers.

A “similarity value” is a number that represents the degree ofsimilarity between two things being compared. For example, a similarityvalue may be a number that indicates the overall similarity between acell sample expression profile using specific phenotype-relatedbiomarkers and a control specific to that template (for instance, thesimilarity to a “deregulated IL-6/STATS signaling pathway” template,where the phenotype is deregulated IL-6/STATS signaling pathway status).The similarity value may be expressed as a similarity metric, such as acorrelation coefficient, or a classification probability or may simplybe expressed as the expression level difference, or the aggregate of theexpression level differences, between a cell sample expression profileand a baseline template.

As used herein, the terms “measuring expression levels,” “obtainingexpression level,” and “detecting an expression level” and the like,includes methods that quantify a gene expression level of, for example,a transcript of a gene, or a protein encoded by a gene, as well asmethods that determine whether a gene of interest is expressed at all.Thus, an assay which provides a “yes” or “no” result without necessarilyproviding quantification, of an amount of expression is an assay that“measures expression” as that term is used herein. Alternatively, ameasured or obtained expression level may be expressed as anyquantitative value, for example, a fold-change in expression, up ordown, relative to a control gene or relative to the same gene in anothersample, or a log ratio of expression, or any visual representationthereof, such as, for example, a “heatmap” where a color intensity isrepresentative of the amount of gene expression detected. The genesidentified as being differentially expressed in tumor cells havingIL-6/STAT3 signaling pathway deregulation may be used in a variety ofnucleic acid or protein detection assays to detect or quantify theexpression level of a gene or multiple genes in a given sample.Exemplary methods for detecting the level of expression of a geneinclude, but are not limited to, Northern blotting, dot or slot blots,reporter gene matrix (see for example, U.S. Pat. No. 5,569,588) nucleaseprotection, RT-PCR, microarray profiling, differential display, 2D gelelectrophoresis, SELDI-TOF, ICAT, enzyme assay, antibody assay,MNAzyme-based detection methods (see U.S. Ser. No. 61/470,919, US2011/0143338; US 2007/0231810; WO WO/2008/122084; WO/2007/041774; andMokany et al., J Am Chem Soc. 2010 January 27; 132(3): 1051-1059, eachof which is incorporated by reference in its entirety), and the like.Optionally a gene whose level of expression is to be detected may beamplified, for example by methods that may include one or more of:polymerase chain reaction (PCR), strand displacement amplification(SDA), loop-mediated isothermal amplification (LAMP), rolling circleamplification (RCA), transcription-mediated amplification (TMA),self-sustained sequence replication (3SR), nucleic acid sequence basedamplification (NASBA), or reverse transcription polymerase chainreaction (RT-PCR). In the preferred embodiment gene expression will bedetected by RT-PCR, preferably using SYBR green.

A “patient” can mean either a human or non-human animal, preferably amammal.

As used herein, “subject”, as refers to an organism or to a cell sample,tissue sample or organ sample derived therefrom, including, for example,cultured cell lines, biopsy, blood sample, or fluid sample containing acell. In many instances, the subject or sample derived therefrom,comprises a plurality of cell types. In one embodiment, the sampleincludes, for example, a mixture of tumor and normal cells. In oneembodiment, the sample comprises at least 10%, 15%, 20%, et seq., 90%,or 95% tumor cells. The organism may be an animal, including but notlimited to, an animal, such as a cow, a pig, a mouse, a rat, a chicken,a cat, a dog, etc., and is usually a mammal, such as a human.

As used herein, the term “pathway” is intended to mean a set of systemcomponents involved in two or more sequential molecular interactionsthat result in the production of a product or activity. A pathway canproduce a variety of products or activities that can include, forexample, intermolecular interactions, changes in expression of a nucleicacid or polypeptide, the formation or dissociation of a complex betweentwo or more molecules, accumulation or destruction of a metabolicproduct, activation or deactivation of an enzyme or binding activity.Thus, the term “pathway” includes a variety of pathway types, such as,for example, a biochemical pathway, a gene expression pathway, and aregulatory pathway. Similarly, a pathway can include a combination ofthese exemplary pathway types.

“IL-6/STAT3 signaling pathway” refers to the intracellular signalingpathway activated when the cytokine IL-6 binds to the IL-6 receptor(IL-6R), and this complex then associates with gp130, inducingdimerization and the initiation of signaling through signal transducerand activator of transcription-3 (STAT3). The IL-6R is composed of twodifferent subunits: (1) an alpha subunit that produces ligandspecificity, and (2) gp130 that is a receptor subunit shared in commonwith other cytokines in the IL-6 family. Binding of IL-6 to its receptorinitiates cellular events including activation of JAK kinases, e.g.,JAK2, and activation of ras-mediated signaling. Activated JAK kinasesphosphorylate and activate STAT transcription factors, i.e., JAK2activates STAT3, that then move into the nucleus to activatetranscription of genes containing STAT3 response elements, e.g., SOCS3.(See, Akira S, et al. Molecular cloning of APRF, a novel IFN-stimulatedgene factor 3 p91-related transcription factor involved in thegp130-mediated signaling pathway. Cell. 1994; 77:63-71; Darnell JE., JrSTATs and gene regulation. Science. 1997; 277:1630-1635; and Starr R, etal., (1997) Nature (London) 387:917-921. The ras-mediated pathway,acting through Shc, Grb-2 and Sos-1 upstream and activating Map kinasesdownstream, activates transcription factors such as ELK-1 and NF-IL-6(CIEBP-beta) that can act through their own cognate response elements inthe genome. These factors and other transcription factors like AP-1 andSRF (serum response factor) that respond to many different signalingpathways come together to regulate a variety of complex promoters andenhancers that respond to IL-6 and other signaling factors. TheIL-6/STAT3 signaling pathway includes, but is not limited to, the genes,and proteins encoded thereby, listed in the Tables in this application.

“IL-6/STAT3 agent” refers to a drug or agent that modulates thecanonical IL-6/STAT3 signaling pathway. An IL-6/STAT3 pathway inhibitorinhibits IL-6/STAT3 pathway signaling. Molecular targets of such agentsmay include JAK2 and STAT3, as well as any of the genes listed herein.Such agents are known in the art and include, but are not limited to:AZD1480 (Hedvat et al., The JAK2 Inhibitor, AZD1480, Potently BlocksStat3 Signaling and Oncogenesis in Solid Tumors. Cancer Cell. 2009 Dec.8; 16(6):487-97), WP1066 (Calbiochem, La Jolla, Calif., USA), NSC 74859,Stattic (Santa Cruz Biotechnology, Inc.; Schust et al., Stattic: A SmallMolecule Inhibitor of STAT3 Activation and Dimerization. Chemistry &Biology. 2006; 13:1235-1242), and LLL12 (Liu et al., Inhinition of STAT3signaling blocks the anti-apoptotic activity of IL-6 in human livercancer cells. J Biol Chem, 285:27429-39, Epub 2010 Jun. 18).

The term “deregulated IL-6/STAT3 pathway” is used herein to mean thatthe IL-6/STAT3 signaling pathway is either hyperactivated orhypoactivated. A IL-6/STAT3 signaling pathway is hyperactivated in asample (for example, a tumor sample) if it has at least 10%, 20%, 30%,40%, 50%, 75%, 100%, 200%, 500%, 1000% greater activity/signaling thanthe IL-6/STAT3 signaling pathway in a normal (regulated) sample. AIL-6/STAT3 signaling pathway is hypoactivated if it has at least 10%,20%, 30%, 40%, 50%, 75%, 100% less activity/signaling in a sample (forexample, a tumor sample) than the IL-6/STAT3 signaling pathway in anormal (regulated) sample. The normal sample with the regulatedIL-6/STAT3 signaling pathway may be from adjacent normal tissue, may beother tumor samples which do not have deregulated IL-6/STAT3 signaling,or may be a pool of samples. Alternatively, comparison of samples'IL-6/STAT3 signaling pathway status may be done with identical sampleswhich have been treated with a drug or agent vs. vehicle. The change inactivation status may be due to a mutation of one or more genes in theIL-6/STAT3 signaling pathway (such as point mutations, deletion, oramplification), changes in transcriptional regulation (such asmethylation, phosphorylation, or acetylation changes), or changes inprotein regulation (such as translation or post-translational controlmechanisms).

The term “oncogenic pathway” is used herein to mean a pathway that whenhyperactivated or hypoactivated contributes to cancer initiation orprogression. In one embodiment, an oncogenic pathway is one thatcontains an oncogene or a tumor suppressor gene.

The term “treating” in its various grammatical forms in relation to thepresent invention refers to preventing (i.e. chemoprevention), curing,reversing, attenuating, alleviating, minimizing, suppressing, or haltingthe deleterious effects of a disease state, disease progression, diseasecausative agent (e.g. bacteria or viruses), or other abnormal condition.For example, treatment may involve alleviating a symptom (i.e., notnecessarily all the symptoms) of a disease of attenuating theprogression of a disease.

“Treatment of cancer,” as used herein, refers to partially or totallyinhibiting, delaying, or preventing the progression of cancer includingcancer metastasis; inhibiting, delaying, or preventing the recurrence ofcancer including cancer metastasis; or preventing the onset ordevelopment of cancer (chemoprevention) in a mammal, for example, ahuman. In addition, the methods of the present invention may bepracticed for the treatment of human patients with cancer. However, itis also likely that the methods would also be effective in the treatmentof cancer in other mammals.

“Treatment of non-cancer inflammatory conditions,” as used herein,refers to partially or totally inhibiting, delaying, or preventing theprogression of the condition; or preventing the onset or development ofthe condition in a mammal, for example, a human. In addition, themethods of the present invention may be practiced for the treatment ofhuman patients with non-cancer inflammatory conditions. However, it isalso likely that the methods would also be effective in the treatment ofthese conditions in other mammals.

As used herein, the term “therapeutically effective amount” is intendedto qualify the amount of the treatment in a therapeutic regimentnecessary to treat cancer and/or non-cancer inflammatory conditions.This includes combination therapy involving the use of multipletherapeutic agents, such as a combined amount of a first and secondtreatment where the combined amount will achieve the desired biologicalresponse. The desired biological response is partial or totalinhibition, delay, or prevention of the progression of cancer, includingcancer metastasis, or partial or total inhibition, delay, or preventionof the progression of a non-cancer inflammatory condition; inhibition,delay, or prevention of the recurrence of cancer including cancermetastasis; or the prevention of the onset of development of cancer(chemoprevention) and/or a non-cancer inflammatory condition in amammal, for example, a human.

“Displaying or outputting a classification result, prediction result, orefficacy result” means that the results of a gene expression basedsample classification or prediction are communicated to a user using anymedium, such as for example, orally, writing, visual display, etc.,computer readable medium or computer system. It will be clear to oneskilled in the art that outputting the result is not limited tooutputting to a user or a linked external component(s), such as acomputer system or computer memory, but may alternatively oradditionally be outputting to internal components, such as any computerreadable medium. Computer readable media may include, but are notlimited to hard drives, floppy disks, CD-ROMs, DVDs, DATs. Computerreadable media does not include carrier waves or other wave forms fordata transmission. It will be clear to one skilled in the art that thevarious sample classification methods disclosed and claimed herein, can,but need not be, computer-implemented, and that, for example, thedisplaying or outputting step can be done by, for example, bycommunicating to a person orally or in writing (e.g., in handwriting).

As noted above the present invention identifies a novel set of genes,i.e., a gene signature, the levels of expression of which in a cellsample may be used to assess the regulation status of IL-6/STATSsignaling pathway in a cell sample or subject. This is significantbecause, prior to Applicants discovery, there were no real time PCRassay based methods available to quantitatively measure IL-6/STATSpathway activity. The gene signature in combination with a companionalgorithm fulfills this need, and provides biomarkers for assessing theIL-6/STATS pathway activity for various applications, e.g.,diagnostic/sample classification, e.g., tumors; predicting treatmentresponse and assigning treatment; determining whether an agent modulatesthe IL-6/STAT3 signaling pathway; and measuring the pharmacodynamiceffect of an agent targeting IL-6/STAT3.

Additionally, due to limitations of cytotoxic based chemotherapies,current oncology drug development is designed to target specificcellular signaling pathways critical for tumor growth and progression.Such targeted drug development requires specific biomarkers to monitorthe activity status of pathway. Compared to more traditional methods,which rely on detecting the expression of one or a few indicators withinthe pathway constituents, multi-gene expression based methods measurepathway alteration as a function of the downstream effect of pathwayregulation on multiple gene expression changes. These downstream geneexpression alterations can potentially capture all changes related toany upstream alteration of a pathway component.

“Disease associated with IL-6/STAT3 dysregulation” refers to a diseaseor condition in which IL-6/STAT3 pathway activity is altered, e.g.,IL-6/STAT3 activity is elevated or decreased relative to a baselinelevel or a non-diseased control sample, and/or a disease or condition inwhich manipulation of IL-6/STAT3 activity may be effective fortreatment. The IL-6/STAT3 pathway has been shown to play a role in theinflammatory response, non-cancer inflammatory conditions, and cancerinitiation and progression. Persistent activation of STAT3 can mediatetumor-promoting inflammation. STAT3 has a dual role in tumorinflammation and immunity by promoting pro-oncogenic inflammatorypathways, including nuclear factor-κB (NF-κB) and IL-6-GP130-JAKpathways, and by opposing STAT1- and NF-κB-mediated T helper 1anti-tumour immune responses. Consequently, STAT3 is a promising targetto redirect inflammation for cancer therapy. (Yu et al. STATs in cancerinflammation and immunity: a leading role for STAT3. Nature Reviews,9:798-809 (2009)). Deregulation of IL6/STAT3 signaling has beenassociated with, e.g., embryonic development, programmed cell death,organogenesis, innate immunity, and adaptive immunity. Activation of theIL-6/STAT3 pathway results in a variety of downstream biologicaleffects, which is reflected by changes in gene expression.

Many cancers and non-cancer inflammatory conditions have been associatedwith aberrant IL-6/STAT3 signaling. Table 1 includes a non-limiting listof exemplary cancers and non-cancer inflammatory conditions that havebeen associated with the IL-6/STAT-3 pathway.

TABLE 1 Exemplary cancers and non-cancer inflammatory conditionsassociated with IL-6/STAT-3. IL6/STAT3 in cancers cancer type evidenceReference multiple Aberrant production of Kawano, M., et al. myelomasIL6 by neoplastic cells Autocrine generation and has been implicated asa requirement of BSF-2/IL- strong contributory factor 6 for humanmultiple to the growth of multiple myelomas. Nature 332: myeloma andother B-cell 83-85, 1988 dyscrasias, T-cell lymphoma, renal and ovariancell carcinomas, and Kaposi sarcoma Stat3, is constitutively Immunity.1999 January; 10(1): 105-15. activated in bone marrow mononuclear cellsfrom patients with multiple myeloma and in the IL-6- dependent humanmyeloma cell line U266 Cholangio- Overexpression of IL6 Meng, F.,Wehbe-Janek, H., Henson, R., cellular reduced MIR370 Smith, H., Patel,T. Epigenetic regulation carcinoma expression and of microRNA-370 byinterleukin-6 in reinstated MAP3K8 malignant human cholangiocytes.expression in malignant Oncogene 27: 378-386, 2008. cholangiocytes invitro and in tumor cell xenografts in vivo. T-cell STAT3 may transformWelte, T., Zhang, S. S. M., Wang, T., lymphomas cells by inducing Zhang,Z., Hesslein, D. G. T., Yin, Z., epigenetic silencing of Kano, A.,Iwamoto, Y., Li, E., Craft, J. E., SHP1 in cooperation with Bothwell, A.L. M., Fikrig, E., Koni, P. A., DNMT1 and HDAC1 in T- Flavell, R. A.,Fu, X.-Y. STAT3 deletion cell lymphomas during hematopoiesis causesCrohn's disease-like pathogenesis and lethality: a critical role ofSTAT3 in innate immunity. Proc. Nat. Acad. Sci. 100: 1879-1884, 2003.malignant expression of C/EBP- Carro, M. S., Lim, W. K., Alvarez, M. J.,glioma beta and STAT3 Bollo, R. J., Zhao, X., Snyder, E. Y., correlatedwith Sulman, E. P., Anne, S. L., Doetsch, F., mesenchymal Colman, H.,Lasorella, A., Aldape, K., differentiation and Califano, A., Iavarone,A. The predicted poor clinical transcriptional network for mesenchymaloutcome transformation of brain tumours. Nature 463: 318-325, 2010. headTGF-alpha/EGFR- J Clin Invest. 1998 Oct. 1; 102(7): 1385-92. and neckmediated autocrine cancer growth of transformed epithelial cells isdependent on activation of Stat3 but not Stat1. leukemia ConstitutiveSTAT Coffer P. J., Koenderman L., de Groot R. activation is present inP. The role of STATs in myeloid many malignancies and differentiationand leukemia. Oncogene, has been especially well 19: 2511-2522, 2000characterized in acute and chronic leukemias Lin T. S., Mahajan S.,Frank D. A. STAT signaling in the pathogenesis and treatment ofleukemias. Oncogene, 19: 2496-2504, 2000 breast Src and JAK familyGarcia R., Bowman T. L., Niu G., Yu H., cancer tyrosine kinases MintonS., Muro-Cacho C. A., Cox C. E., cooperate to mediate Falcone R.,Fairclough R., Parsons S., constitutive Stat3 Laudano A., Gazit A.,Levitzki A., Kraker activation in the absence A., Jove R. Constitutiveactivation of Stat3 of EGF stimulation in by the Src and JAK tyrosinekinases model human breast participates in growth regulation of humancancer cell lines breast carcinoma cells. Oncogene, 20: 2499-2513, 2001.renal cell activated STAT3 Clin Cancer Res. 2002 April; 8(4): 945-54.carcinoma melanoma activated STAT3 Clin Cancer Res. 2002 April; 8(4):945-54. ovarian activated STAT3 Clin Cancer Res. 2002 April; 8(4):945-54. carcinoma lung cancer activated STAT3 Clin Cancer Res. 2002April; 8(4): 945-54. prostate cancer activated STAT3 Clin Cancer Res.2002 April; 8(4): 945-54. pancretic activated STAT3 Clin Cancer Res.2002 April; 8(4): 945-54. adenocarcinoma non-cancer inflammatorycondition type Evidence Reference hypoferremia of IL6 is the necessaryand Nemeth, E., Rivera, S., Gabayan, V., inflammation sufficientcytokine for the Keller, C., Taudorf, S., Pedersen, B. K., induction ofhepcidin Ganz, T. IL-6 mediates hypoferremia of during inflammationinflammation by inducing the synthesis of the iron regulatory hormonehepcidin. J. Clin. Invest. 113: 1271-1276, 2004. acute-phase IL6regulates the zinc Liuzzi, J. P., Lichten, L. A., Rivera, S., responseto importer ZIP14 and Blanchard, R. K., Aydemir, T. B., Knutson,inflammation contributes to the M. D., Ganz, T., Cousins, R. J.Interleukin- and infection hypozincemia 6 regulates the zinc transporterZip14 in accompanying the acute- liver and contributes to thehypozincemia phase response to of the acute-phase response. Proc. Nat.inflammation and Acad. Sci. 102: 6843-6848, 2005. infection productionof both Alonzi, T., D. Maritano, B. Gorgoni, G. proinflammatory andRizzuto, C. Libert, V. Poli. 2001. Essential antiinflammatory role ofSTAT3 in the control of the acute- cytokines was increased phaseresponse as revealed by inducible and prolonged, probably geneinactivation in the liver. Mol. Cell. as a result of STAT3 Biol. 21:1621 deletion in macrophages. chronic constitutive activation of Hanada,T., T. Yoshida, I. Kinjyo, S. inflammation Stat3 has also beenMinoguchi, H. Yasukawa, S. Kato, H. observed in chronic Mimata, Y.Nomura, Y. Seki, M. Kubo, A. inflammation Yoshimura. 2001. A mutant formof JAB/SOCS1 augments the cytokine- induced JAK/STAT pathway byaccelerating degradation of wild-type JAB/CIS family proteins throughthe SOCS-box. J. Biol. Chem. 276: 40746 inflammation in reducedinflammation, as Dai, J., Miller, A. H., Bremner, J. D., cardiovascularmeasured by IL6 level, is Goldberg, J., Jones, L., Shallenberger, L., animportant mechanism Buckham, R., Murrah, N. V., Veledar, E., linking theMediterranean Wilson, P. W., Vaccarino, V. Adherence to diet to reducedthe Mediterranean diet is inversely cardiovascular risk associated withcirculating interleukin-6 among middle-aged men. Circulation 117:169-175, 2008. STAT3 is crucial in Jacoby, J. J., Kalinowski, A., Liu,M.-G., cardiomyocyte resistance Zhang, S. S.-M., Gao, Q., Chai, G.-X.,Ji, to inflammation and other L., Iwamoto, Y., Li, E., Schneider, M.,acute injury and in the Russell, K. S., Fu, X.-Y. Cardiomyocyte-pathogenesis of age- restricted knockout of STAT3 results in relatedheart failure. higher sensitivity to inflammation, cardiac fibrosis, andheart failure with advanced age. Proc. Nat. Acad. Sci. 100: 12929-12934, 2003. systemic juvenile serum IL6 concentration Rooney, M.,David, J., Symons, J., Di rheumatoid rises significantly in Giovine, F.,Varsani, H., Woo, P. arthritis conjunction with the fever Inflammatorycytokine responses in spike associated with juvenile chronic arthritis.Brit. J. systemic juvenile Rheumatol. 34: 454-460, 1995. rheumatoidarthritis Staphylococcus Il6-mediated T-cell McLoughlin, R. M., Jenkins,B. J., Grail, epidermidis- recruitment required D., Williams, A. S.,Fielding, C. A., Parker, induced peritoneal gp130-dependent Stat3 C. R.,Ernst, M., Topley, N., Jones, S. A. inflammation activation IL-6trans-signaling via STAT3 directs T cell infiltration in acuteinflammation. Proc. Nat. Acad. Sci. 102: 9589-9594, 2005. pulmonary IL-6induction of lung Am J Physiol Lung Cell Mol Physiol. 2012 inflammationinflammation occurs via April; 302(7): L627-39. Epub 2012 Jan. 20. Stat3

By utilizing IL-6 stimulation and siRNA mediated STAT3 knockdown inHepG2 and MCF10A cells followed by gene expression profiling analysis,the inventors have identified a list of 88 response genes whoseexpression was upregulated in response to IL-6 and whose increasedexpression levels were diminished by treatment with STAT3 siRNA. These88 IL-6/STAT3 response genes were further evaluated by real-time PCRwith 16 samples from a panel of 13 different cell lines eitherstimulated with IL-6 and/or inhibited with STAT3 siRNA. Sixteen (16)genes were identified as a specific panel of indicators for IL-6/STAT3pathway regulation using a random forest classifier method. The 16 genesignature predicted the regulatory status of IL-6/STAT3 pathway in atraining set of 16 samples with an accuracy of 87.5% during crossvalidation process with a Random Forest method. Therefore, the inventorshave verified that they have identified a novel gene expressionsignature comprising a specific set of 16 genes, the expression of whichmay be assayed (preferably by RT-PCR) to monitor the regulatory statusof IL-6/STAT3 pathway activity, and related applications involving themodulation of this important signaling pathway.

In particular, the inventors discovered that the 16 genes listed belowprovide a gene expression signature to assess the regulatory status ofthe IL-6/STAT3 pathway, e.g., differentially classify positiveregulation of the IL-6/STAT3 pathway from negative regulation of theIL-6/STAT3 pathway.

STAT3 SOCS3 IFITM2 CEBPD JUNB TUBB2A IL6ST CASP4 PROS1 TNERSF1A PVRL2PHF21A BCL3 NRP1 GLRX TGM2

Based on these results, the invention provides methods and materials forassaying the IL-6/STAT3 pathway activity level, e.g., in real time, byassaying the expression levels of these 16 genes or a subset thereofalone or in combination with other genes that are involved in thispathway. Preferably the gene subset assayed will comprise at least 5 ofthese genes, more preferably at least 10 of the genes, most preferablyall of these 16 genes. Exemplary primers for amplification of the 16IL-6/STAT3 signature genes are described in the application (seeExperimental Section and FIG. 6). Of course alternative primers may beused and indeed may be required, if e.g., the assay measures expressionof orthologs of the listed genes, e.g., rodent orthologs such as inanimal assays designed to assess drug efficacy or side effects.

As disclosed in detail in the Experimental Section, this gene expressionsignature has been developed from cell lines in response to specificpathway manipulation with microarray analysis. Few previous studies haveverified their signature genes in terms of different cell lines andreal-time PCR platform. Therefore, by developing and verifying a uniquegene expression signature correlated to the activation of the IL-6/STAT3pathway with an companion algorithm, the inventors provide a novel andimproved workflow for quantitative pathway gene expression signature forthe identification and verification of cells and samples wherein thispathway is affected using a real-time PCR platform.

The inventive gene expression signature, because of the manner by whichit was determined, should accurately reflect the regulatory status ofIL-6/STAT3 pathway activity and be useful in different assays such asscreening for compounds that modulate IL-6/STAT3 signaling and foridentifying cells wherein IL-6/STAT3 signaling is abnormal as inmalignancy.

As discussed above, and in detail in the Experimental Section, thepresent inventors identified this signature gene set from an initiallist of 88 IL-6/STAT3 response genes identified with microarray analysisin HepG2 and MCF10A cells treated with IL-6 and STAT3-targeting siRNA.The IL-6/STAT3 response genes were validated with real-time PCR in atraining set of 16 samples, and 16 IL-6/STAT3 signature genes wereidentified by a random forest method.

The accuracy and predictive value of this 16 gene signature was laterverified by cross validation in those 16 samples using real-time PCR, inwhich 9 samples were stimulated with IL-6 (“positively regulated”) andthe 7 samples were transfected with STAT3 siRNA (“negativelyregulated”). As shown infra and in the Figures referenced in theExperimental Section, this 16 gene signature had an accuracy of 87.5% inpredicting the regulatory status of IL-6/STAT3 pathway activity in these16 samples. Therefore, the 16 gene signature and the genes in thissignature may be used as biomarkers for monitoring the regulatory statusof IL-6/STAT3 pathway activity.

In a preferred embodiment, the expression of these 16 genes may bedetermined in samples by microarray and/or RT-PCR. In an especiallypreferred embodiment, the expression of these 16 genes may be determinedby use of SYBR Green based real-time PCR, the gene expression dataanalyzed by ΔΔCt method, and the pathway activity determined with therandom forest method.

In these methods the regulatory status of a cell sample may bedetermined by comparing the expression profile of one or more of these16 genes, preferably at least 5 of these genes, to control samples(e.g., cells having a normal IL-6/STAT3 pathway activity). The assayedcell sample for which regulatory status may be evaluated according tothe invention may comprise any cell or cell sample wherein IL-6/STATSpathway activity is desirably assayed. This includes by way of examplepotentially malignant cells, cells which have been obtained from apatient subjected to a chemotherapy regimen which potentially affectsIL-6/STAT3 pathway activity, cells wherein IL-6/STAT3 pathwayderegulation status is desirably evaluated in a sample; cell sampleswhich are to be classified as having a deregulated or regulatedIL-6/STAT3 signaling pathway; a cell sample wherein it is to bedetermined whether an agent modulates the IL-6/STAT3 signaling pathwayin sample; and the like. In addition, the present signature andbiomarkers comprised therein can be used to predict the response of asubject to an agent that modulates the IL-6/STAT3 signaling pathway;assigning treatment to a subject; and evaluating the pharmacodynamiceffects of therapies designed to regulate IL-6/STAT3 pathway signaling.

Because the present invention relies upon a comparison of the levels ofexpression by different genes in cell samples, practice of the inventiontypically requires control and treatment samples to determine therelative regulatory status of a target cell sample vs control. Thetarget cell sample, e.g., may be one manipulated by different means thatmay affect IL-6/STAT3 pathway regulation status such as contacting withsiRNA(s), drug treatment and the like and the control will be theappropriate control for that manipulation. For example the control cellswill be treated identically (culture conditions, time, excipients,vehicles) except for the absence of the particular tested manipulationagent such as exposure to a chemotherapeutic agent. Alternatively, thecontrol sample may be computer generated random ΔΔCT variation for eachgene.

In the present invention, target polynucleotide molecules are typicallyextracted from a sample taken from an individual afflicted with canceror tumor cell lines, and corresponding normal/control tissues or celllines, respectively. Samples may also be taken from primary cell linesor ex vivo cultures of cells taken from an animal or patient. The samplemay be collected in any clinically acceptable manner, but must becollected such that biomarker-derived polynucleotides (i.e., RNA) arepreserved. mRNA or nucleic acids derived therefrom (i.e., cDNA oramplified DNA) are preferably labeled distinguishably from standard orcontrol polynucleotide molecules, and both are simultaneously orindependently hybridized to a microarray comprising some or all of thebiomarkers or biomarker sets or subsets described above. Alternatively,mRNA or nucleic acids derived therefrom may be labeled with the samelabel as the standard or control polynucleotide molecules, wherein theintensity of hybridization of each at a particular probe is compared. Asample may comprise any clinically relevant tissue sample, such as atumor biopsy, fine needle aspirate, or hair follicle, or a sample ofbodily fluid, such as blood, plasma, serum, lymph, ascitic fluid, cysticfluid, urine. The sample may be taken from a human, or, in a veterinarycontext, from non-human animals such as ruminants, horses, swine orsheep, or from domestic companion animals such as felines and canines.Additionally, the samples may be from frozen or archived formalin-fixed,paraffin-embedded (H-PE) tissue samples.

Methods for preparing total and poly(A)+RNA are well known and aredescribed generally in Sambrook et al., MOLECULAR CLONING—A LABORATORYMANUAL (2ND ED.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. (1989)) and Ausubel et al., CURRENT PROTOCOLS IN MOLECULARBIOLOGY, vol. 2, Current Protocols Publishing, New York (1994)).

RNA may be isolated from eukaryotic cells by procedures that involvelysis of the cells and denaturation of the proteins contained therein.Cells of interest include wild-type cells (i.e., non-cancerous),drug-exposed wild-type cells, tumor- or tumor-derived cells, modifiedcells, normal or tumor cell line cells, and drug-exposed modified cells.

Additional steps may be employed to remove DNA. Cell lysis may beaccomplished with a nonionic detergent, followed by microcentrifugationto remove the nuclei and hence the bulk of the cellular DNA. In oneembodiment, RNA is extracted from cells of the various types of interestusing guanidinium thiocyanate lysis followed by CsCl centrifugation toseparate the RNA from DNA (Chirgwin et al., Biochemistry 18:5294-5299(1979)). Poly(A)+RNA is selected by selection with oligo-dT cellulose(see Sambrook et al, MOLECULAR CLONING—A LABORATORY MANUAL (2ND ED.),Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.(1989). Alternatively, separation of RNA from DNA can be accomplished byorganic extraction, for example, with hot phenol orphenol/chloroform/isoamyl alcohol. If desired, RNase inhibitors may beadded to the lysis buffer. Likewise, for certain cell types, it may bedesirable to add a protein denaturation/digestion step to the protocol.

For many applications, it is desirable to preferentially enrich mRNAwith respect to other cellular RNAs, such as transfer RNA (tRNA) andribosomal RNA (rRNA). Most mRNAs contain a poly(A) tail at their 3′ end.This allows them to be enriched by affinity chromatography, for example,using oligo(dT) or poly(U) coupled to a solid support, such as celluloseor Sephadex. (see Ausubel et al., CURRENT PROTOCOLS IN MOLECULARBIOLOGY, vol. 2, Current Protocols Publishing, New York (1994). Oncebound, poly(A)+mRNA is eluted from the affinity column using 2 mMEDTA/0.1% SDS.

The sample of RNA can comprise a plurality of different mRNA molecules,each different mRNA molecule having a different nucleotide sequence. Ina specific embodiment, the mRNA molecules in the RNA sample comprise atleast 100 different nucleotide sequences. More preferably, the mRNAmolecules of the RNA sample comprise mRNA molecules corresponding toeach of the biomarker genes. In another specific embodiment, the RNAsample is a mammalian RNA sample.

In a specific embodiment, total RNA or mRNA from cells is used in themethods of the invention. The source of the RNA can be cells of a plantor animal, human, mammal, primate, non-human animal, dog, cat, mouse,rat, bird, yeast, eukaryote, prokaryote, etc. In specific embodiments,the method of the invention is used with a sample containing total mRNAor total RNA from 1×10⁶ cells or less. In another embodiment, proteinscan be isolated from the foregoing sources, by methods known in the art,for use in expression analysis at the protein level.

Probes to the homologs of the biomarker sequences disclosed herein canbe employed preferably wherein non-human nucleic acid is being assayed.

In a preferred embodiment of the invention, the IL-6/STAT3 pathwayregulation status will be determined based on the expression levels ofall of the 16 genes in the IL-6/STAT3 pathway signature versus thecontrol sample. However, it is envisioned that IL-6/STAT3 pathwayregulation status may also be determined by assaying the expression of asubset of these 16 genes or biomarkers, i.e., any combination of atleast 2 of these genes, at least 3 of these genes, at least 4 of thesegenes, at least 6 of these genes, at least 7 of these genes, . . . orall of these 16 genes. In addition, it is within the scope of theinvention to further assay the expression of additional genes whichaffect and/or correlate to IL-6/STAT3 pathway regulation status.

Therefore, one aspect of the invention provides a set of 16 biomarkersor a subset thereof whose expression is correlated with IL-6/STAT3signaling pathway deregulation. These biomarkers identified as usefulfor classifying subjects according to regulation status of theIL-6/STAT3 signaling pathway may also be used for classification of cellsamples, including but not limited to tumors, by assessing pathwayactivation status; predicting response to treatment, i.e., prospectivelyidentifying patients harboring tumors that have high levels of aparticular pathway activity before treating the patients with inhibitorstargeting the pathway; assigning treatment; and as early efficacybiomarkers, i.e., an early readout of efficacy. A gene expressionsignature for pathway activity may also be used to screen for agentsthat modulate the IL-6/STAT3 signaling pathway. Furthermore, geneexpression signatures for pathway activation may also be used aspharmacodynamic biomarkers, i.e., monitoring pathway inhibition inpatient tumors or peripheral tissues post-treatment.

Another aspect of the invention provides a method of using thesebiomarkers or a microarray containing to distinguish tumor types indiagnosis or to predict response to therapeutic agents.

Yet other aspects of the invention provide methods of using thesebiomarkers or a microarray containing as pharmacodynamic biomarkers,i.e. monitoring pathway inhibition in patient tumors or peripheraltissues post-treatment; as response prediction biomarkers, i.e.,prospectively identifying patients harboring tumors that have highlevels of a particular pathway activity before treating the patientswith inhibitors targeting the pathway; and as early efficacy biomarkers,i.e., an early readout of efficacy.

In another embodiment, the invention provides a set of 16 biomarkers ora subset thereof, or a microarray containing them, that can be used topredict response of a subject to a IL-6/STATS signaling pathway agent.In a more specific embodiment, the invention provides a subset of thedisclosed set of 16 biomarkers that can be used to predict the responseof a subject to an agent that modulates the IL-6/STAT3 signalingpathway. In another embodiment, the invention provides a set of 16biomarkers that can be used to select a IL-6/STAT3 pathway agent fortreatment of a subject with cancer and/or a non-cancer inflammatorycondition, e.g., hypoferremia of inflammation, acute-phase response toinflammation and infection, chronic inflammation, inflammation incardiovascular, systemic juvenile rheumatoid arthritis, Staphylococcusepidermidis-induced peritoneal inflammation, and pulmonary inflammation.In yet another embodiment, the pulmonary inflammation condition includesadult respiratory distress syndrome, shock lung, chronic pulmonaryinflammatory disease, pulmonary sarcoidosis, pulmonary fibrosis andsilicosis. In a more specific embodiment, the invention provides a setof 16 biomarkers that can be used to select a IL-6/STAT3 pathway agentfor treatment of a subject with cancer, e.g., lung, breast, esophageal,head and neck, colonic, gastrointestinal, prostate, multiple myeloma,hepatic, ovarian, neuroblastoma, glioblastoma, melanoma, pancreaticadenocarcinoma, renal cell carcinoma, cholangiocellular carcinoma, andvarious leukemias and lymphomas. Non-limiting examples thereof includelow grade/follicular non-Hodgkin's lymphoma (NHL), small lymphocytic(SL) NHL, intermediate grade/follicular NHL, intermediate grade diffuseNHL, chronic lymphocytic leukemia (CLL), high grade immunoblastic NHL,high grade lymphoblastic NHL, high grade small noncleaved cell NHL,bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma,Waldenstrom's Macroglobulinemia and T cell lymphomas and leukemias.Alternatively, these biomarkers can be used to predict response of asubject to a IL-6/STAT3 signaling pathway agent or to select aIL-6/STAT3 signaling pathway agent for treatment of a subject with anon-cancer inflammatory condition, e.g., hypoferremia of inflammation,acute-phase response to inflammation and infection, chronicinflammation, inflammation in cardiovascular, systemic juvenilerheumatoid arthritis, Staphylococcus epidermidis-induced peritonealinflammation, and pulmonary inflammation. In one embodiment, thepulmonary inflammation condition includes adult respiratory distresssyndrome, shock lung, chronic pulmonary inflammatory disease, pulmonarysarcoidosis, pulmonary fibrosis and silicosis. Additionally, thebiomarkers can be used to detect inflammation sites in vivo or ex vivo.

In another embodiment, the invention provides a set of 16 geneticbiomarkers or a subset thereof, or a microarray containing them, thatcan be used to determine whether an agent has a pharmacodynamic effecton the IL-6/STAT3 signaling pathway in a subject. The biomarkersprovided may be used to monitor inhibition of the IL-6/STAT3 signalingpathway at various time points following treatment of a subject withsaid agent. In a more specific embodiment, the invention provides asubset of the disclosed 16 biomarkers that can be used to monitorpharmacodynamic activity of an agent on the IL-6/STAT3 signalingpathway.

The subject biomarkers may be used alone or in combination withbiomarkers outside the set. For example, biomarkers that distinguishIL-6/STAT3 pathway regulation status may be used in combination withbiomarkers that distinguish growth factor signaling pathway regulationstatus. Any of the biomarker sets provided herein also may also be usedin combination with other biomarkers for cancer, inflammation, or forany other clinical or physiological condition.

As noted in a preferred embodiment, the expression value of all 16 genesis assayed by realtime PCR to determine the IL-6/STAT3 pathwayregulatory status. To ensure accuracy the expression value of these 16genes plus control genes (i.e., 1 or more house keeping genes, e.g., 5house keeping genes) is measured on both the control cell sample and thetreatment sample and the ΔΔCt is calculated. This ΔΔCt value of those 16genes is then compared to ΔΔCt value of 16 genes in a training data poolthat contains 16 samples (7 negatively regulated and 9 positivelyregulated in terms of pathway activity). In the exemplified embodiments,the random forest method is used to determine the regulatory status ofthe particular target cell sample.

The present invention further provides kits and kit components foreffecting the subject gene expression assay methods. In a preferredexemplary embodiment, the kit will comprise an IL-6/STAT3 signaling PCRarray product comprising one or more sequences corresponding to these 16genes, preferably all 16 of these genes or the majority thereof. Theinvention further may preferably include a web based system for analysisof the gene expression data.

The present invention further provides compositions for the detection ofthe gene signature comprising 16 genes, or a subset thereof, and the usethereof in determining the regulation status of the IL-6/STAT3 signalingpathway in a cell sample or subject. The composition may furthercomprise primers for the amplification of between 1 and 10 housekeepinggenes, e.g., 5 housekeeping genes. In one embodiment, the compositionscomprise primers that are in contact with the sample to be tested forIL-6/STAT3 pathway activity. Such primers may have comprises afluorophore to provide for a qualitative and/or quantitative readout ofthe amplification reaction. In one embodiment, at least one primercomprises a fluorophore and matched fluorescence quencher.

For example, the composition comprises primers that amplify at least 2genes selected from the group consisting of STAT3, SOCS3, IFITM2, CEBPD,JUNB, TUBB2A, IL-6ST, CASP4, PROS1, TNFRSFIA, PVRL2, PHF21A, BCL3, NRP1,GLRX, and TGM2, or an ortholog or variant thereof, which allow fordetection of IL-6/STAT3 pathway activation in a cell sample or subject.The enumerated genes in the 16 gene signature correspond to thefollowing accession numbers: NM_(—)213662, NM_(—)003955, NM_(—)006435,NM_(—)005195, NM_(—)002229, NM_(—)001069, NM_(—)002184, NM_(—)001225,NM_(—)000313, NM_(—)001065, NM_(—)002856, NM_(—)016621, NM_(—)005178,NM_(—)003873, NM_(—)002064, and NM_(—)004613. The composition maycomprise at least five of the following primer pairs:

TGACATGGAGTTGACCTCG and CTGGAACCACAAAGTTAGTAGTTTC; CCACCTACTGAACCCTCCTCCand TCTTCCGACAGAGATGCTGAA; TCCCACGTACTCTATCTTCCATTC andCTGATGCAGGACTCGGCTG; CGCCATGTACGACGACGAG and CGCCTTGTGATTGCTGTTG;CGACTACAAACTCCTGAAACCG and GAAGAGGCGAGCTTGAGAGAC; AACTTCTCAGATCAATCGTGCand AGACCATGCTTGAGGACAAC; AAGATTTGAAACAGTTGGCATGGAG andCCTTCACTGAGGCATGTAGC; GAGAGACAGCACAATGGGCTC andCTTCCGAAATACTTCCTCTAGGTG; ATCGGATACAGGCCCTAAGTC andTTGTCCAAGACGGCAAGTTG; TGTTACACTAATAGAAACTTGGCAC andCCTTAGGACAGTTCAGCTTGC; AAGCCAAAGAGACTCAGGTG and CAGGTATCAGGGCTGGTTCCTC;GGCAGAAGGAGATGCACAGC and TCAGAGTCTACAGGTTTGGAGAG;CACTCTCTACCAGATAACTGAGGAG and TAATAATTTACATCGTGATCCGTGC;CAACAACTATGATACACCTGAGC and TTCCACTTCACAGCCCAGC; GCAGAGGCTGTGGTCATGC andTGCTTTAATCTTTGCTGGTAGTC; and CTTCACAAGGGCGAACCAC andGCGGCAGACGTACTCCTCAG.

In one embodiment, the composition includes primers for amplification ofat least 5, at least 6, at least 7, at least 8, at least 9, at least 10,at least 11, at least 12, at least 13, at least 14, at least 15, or atleast 16 of said genes. Preferably, the composition includes primers foramplification of at least 10 to 15 of said genes. More preferably, thecomposition includes primers for amplification of all 16 said genes.

In addition to primer pairs for the amplification of Notch signatureprofiel genes, the composition may further comprise a DNA or RNApolymerase. In one embodiment, the compositions of the invention areadapted for effecting PCR, real-time PCR, strand displacementamplification (SDA), loop-mediated isothermal amplification (LAMP),rolling circle amplification (RCA), transcription-mediated amplification(TMA), self-sustained sequence replication (3SR), nucleic acid sequencebased amplification (NASBA), reverse transcriptase polymerase chainreaction (RT-PCR), or helicase-dependent isothermal DNA amplification.

Real time PCR, also abbreviated as Q-PCR, qPCR, QRT-PCR, or RT-qPCR, isa laboratory technique based on the PCR (polymerase chain reaction), toamplify and simultaneously quantify targeted DNA molecules, which aremost often produced by reverse transcription in order to detect andquantify the template mRNA. It enables both detection and quantification(as absolute copy numbers or relative amount of reference genes) of oneor more specific sequences in a DNA sample. The procedure follows thegeneral principle of polymerase chain reaction. The amplified DNA isdetected as the reaction progresses in real time. Two common methods fordetection of products in real-time PCR are: (1) sequence-specific DNAprobes consisting of oligonucleotides that are labeled with afluorescent reporter which permits detection only after hybridization ofthe probe with its complementary DNA target, and (2) non-specificfluorescent dyes that intercalate with any double-stranded DNA. Thecommonly used reagent for method (1) is TaqMan probes and for method (2)is the SYBR Green I dye. Frequently, real-time PCR is combined withreverse transcription to quantify RNA (including messenger RNA andNon-coding RNA).

TaqMan probes are hydrolysis probes that are designed to increase thespecificity of real-time PCR assays (Holland, P M; Abramson, RD; Watson,R; Gelfand, DH (1991). “Detection of specific polymerase chain reactionproduct by utilizing the 5′-3′ exonuclease activity of Thermus aquaticusDNA polymerase”. Proceedings of the National Academy of Sciences of theUnited States of America 88 (16): 7276-80. PMID 1871133; Gelfand, etal., U.S. Pat. No. 5,210,015; Mayrand; Paul E.: U.S. Pat. No.7,413,708). TaqMan utilizes a dual-labeled probe (containing afluorophore and matched fluorescence quencher) and fluorophore-baseddetection. During hybridization to the complementary target sequence,the 5′-3′ nuclease activity of Taq DNA polymerase releases thefluorophore from proximity to the quencher, generating fluorescenceintensity proportionate to the amount of complementary target sequencein the reaction. As in other real-time PCR methods, the resultingfluorescence signal permits quantitative measurements of theaccumulation of the product during the exponential stages of the PCR;however, the TaqMan probe significantly increases the specificity of thedetection.

TaqMan probes consist of a fluorophore covalently attached to the 5′-endof the oligonucleotide probe and a quencher at the 3′-end. Severaldifferent fluorophores (e.g. 6-carboxyfluorescein, acronym: FAM, ortetrachlorofluorescin, acronym: TET) and quenchers (e.g.tetramethylrhodamine, acronym: TAMRA, or dihydrocyclopyrroloindoletripeptide minor groove binder, acronym: MGB) are available. Thequencher molecule quenches the fluorescence emitted by the fluorophorewhen excited by the cycler's light source via FRET (FluorescenceResonance Energy Transfer). As long as the fluorophore and the quencherare in proximity, quenching inhibits any fluorescence signals.

TaqMan probes are designed such that they anneal within a DNA regionamplified by a specific set of primers. As the Taq DNA polymeraseextends the primer and synthesizes the nascent strand, the 5′ to 3′exonuclease activity of the polymerase degrades the probe that hasannealed to the template. Degradation of the probe releases thefluorophore from it and breaks the close proximity to the quencher, thusrelieving the quenching effect and allowing fluorescence of thefluorophore. Hence, fluorescence detected in the real-time PCR thermalcycler is directly proportional to the fluorophore released and theamount of DNA template present in the PCR.

Another commonly used reagent for detection of products in real-time PCRis SYBR Green I (SG), an asymmetrical cyanine dye that is also used as anucleic acid stain in molecular biology. SYBR Green I binds todouble-stranded DNA. The resulting DNA-dye-complex absorbs blue light(λmax=488 nm) and emits green light (λmax=522 nm). SYBR Green I can bereadily used for real-time PCR detection because there is a linearrelation between the double-stranded DNA synthesized and the amount ofgreen light emitted.

Reagents for detection of products in real-time PCR also include doublestrand nucleic acid specific dyes such as SYBR Gold, ethidium bromide,propidium bromide, Pico Green, reagents for detection of real-time PCRproducts include the fluorescent dyes and quenchers listed in Table 1below. Hoechst 33258, YO-PRO-I and YO-YO-I, Boxto, Evagreen, LC Green,LC Green Plus and Syto 9.

Additional exemplary

TABLE 1 Exemplary fluorescent dyes and compatible quenchers suitable fordetection of real time PCR products. Abbreviations: HEX:6-carboxy-2′,4,4′,5′,7,7′-hexachlorofluorescein, succinimidyl ester;6-FAM: 6-carboxyfluorescein; ROX: 6-ROXN (6-carboxy-X-rhodamine); BHQ-1:Black Hole Quencher 1, Biosearch Technologies, Inc., Novato, CA; BHQ-2:Black Hole Quencher 2, Biosearch Technologies, Inc., Novato, CA; BHQ-3:Black Hole Quencher 3, Biosearch Technologies, Inc., Novato, CA. MaxExci- Max Emis- Compatible Dye tation (nm) sion (nm) Quencher(s) 6-FAM494 515 BHQ-1, TAMRA JOE 520 548 BHQ-1, TAMRA TET 521 536 BHQ-1, TAMRACal Fluor Gold 540 520 548 BHQ-1 HEX 535 555 BHQ-1, TAMRA Cal FluorOrange 560 522 541 BHQ-1 TAMRA 555 576 BHQ-2 Cy3 550 570 BHQ-2 Quasar570 548 566 BHQ-2 Cal Fluor Red 590 565 588 BHQ-2 ROX 573 602 BHQ-2Texas Red 583 603 BHQ-2 Cy5 651 674 BHQ-3 Quasar 670 647 667 BHQ-3 Cy5.5675 694 BHQ-3

TaqMan requires producing double-labeled probes specific for eachproduct, which can increase the cost of TaqMan-based real-time PCRsystem. However, unlike SYBR Green I, TaqMan can readily be utilized formultiplex PCR since a reaction can contain multiple TaqMan probes, eachspecific for a particular amplicon and each utilizing a distinguishablefluorophore.

In addition, biomarker expression levels may be determined using amicroarray, optionally together with amplification of sample nucleicacids (e.g., as described in the preceding paragraphs). A number ofdifferent array configurations and methods of their production are knownto those skilled in the art (see, for example, U.S. Pat. Nos. 5,445,934;5,532,128; 5,556,752; 5,242,974; 5,384,261; 5,405,783; 5,412,087;5,424,186; 5,429,807; 5,436,327; 5,472,672; 5,527,681; 5,529,756;5,545,531; 5,554,501; 5,561,071; 5,571,639; 5,593,839; 5,599,695;5,624,711; 5,658,734; and 5,700,637, each of which is herebyincorporated by reference in its entirety). Microarray technology allowsfor the measurement of the steady-state level of large numbers ofpolynucleotide sequences simultaneously. Microarrays currently in wideuse include cDNA arrays and oligonucleotide arrays.

cDNA microarrays consist of multiple (e.g., thousands) of differentcDNAs spotted (e.g., using a robotic spotting device) onto knownlocations on a solid support, such as a glass microscope slide, ontowhich the probes are covalently or non-covalently attached. The cDNAsare typically obtained by PCR amplification of plasmid library insertsusing primers complementary to the vector backbone portion of theplasmid or to the gene itself for genes where sequence is known. PCRproducts suitable for production of microarrays are typically between0.5 and 2.5 kB in length. In a typical microarray experiment, RNA(either total RNA or poly A RNA) is isolated from cells or tissues ofinterest and is reverse transcribed to yield cDNA. Labeling is usuallyperformed during reverse transcription by incorporating a labelednucleotide in the reaction mixture. A microarray is then hybridized withlabeled RNA, and relative expression levels calculated based on therelative concentrations of cDNA molecules that hybridized to the cDNAsrepresented on the microarray. Microarray analysis can be performed bycommercially available equipment, following manufacturer's protocols,such as, e.g., by using Affymetrix GeneChip® technology, AgilentTechnologies cDNA microarrays, Illumina Whole-Genome DASL® array assays,or any other comparable microarray technology.

Probes capable of hybridizing to one or more biomarker RNAs or cDNAs maybe attached to the substrate at a defined location (“addressablearray”). Probes can be attached to the substrate in a wide variety ofways, as will be appreciated by those in the art. In some embodiments,the probes are synthesized first and subsequently attached to thesubstrate. In other embodiments, the probes are synthesized on thesubstrate. In some embodiments, probes are synthesized on the substratesurface using techniques such as photopolymerization andphotolithography.

In some embodiments, microarrays are utilized in a RNA-primed,Array-based Klenow Enzyme (“RAKE”) assay. See Nelson, P. T. et al.(2004) Nature Methods 1(2):1-7; Nelson, P. T. et al. (2006) RNA12(2):1-5, each of which is incorporated herein by reference in itsentirety. In these embodiments, total RNA is isolated from a sample.Optionally, small RNAs can be further purified from the total RNAsample. The RNA sample is then hybridized to DNA probes immobilized atthe 5′-end on an addressable array. The DNA probes comprise a basesequence that is complementary to a target RNA of interest, such as oneor more biomarker RNAs capable of specifically hybridizing to a nucleicacid comprising a sequence that is identically present in one of thegenes of interest under standard hybridization conditions.

Analyses using microarrays are generally based on measurements of theintensity of the signal received from a labeled probe used to detect acDNA sequence from the sample that hybridizes to a nucleic acid probeimmobilized at a known location on the microarray (see, for example,U.S. Pat. Nos. 6,004,755; 6,218,114; 6,218,122; and 6,271,002). Forexample, fluorescently labeled cDNA probes may be generated throughincorporation of fluorescently labeled deoxynucleotides by reversetranscription of RNA extracted from the cells of interest.Alternatively, the RNA may be amplified by in vitro transcription andlabeled with a marker, such as biotin. The labeled probes are thenhybridized to the immobilized nucleic acids on the microchip underhighly stringent conditions. After stringent washing to remove thenon-specifically bound probes, the chip is scanned by confocal lasermicroscopy or by another detection method, such as a CCD camera. The rawfluorescence intensity data in the hybridization files are generallypreprocessed with the robust multichip average (RMA) algorithm togenerate expression values. Array-based gene expression methods areknown in the art and have been described in numerous scientificpublications as well as in patents (see, for example, M. Schena et al.,Science, 1995, 270: 467-470; M. Schena et al., Proc. Natl. Acad. Sci.USA 1996, 93: 10614-10619; J. J. Chen et al., Genomics, 1998, 51:313-324; U.S. Pat. Nos. 5,143,854; 5,445,934; 5,807,522; 5,837,832;6,040,138; 6,045,996; 6,284,460; and 6,607,885).

In one embodiment, the primers are contained in one or more wells of amulti-well reaction vessel. In another embodiment, the primers foramplification of at least two of said genes are included together in aduplex or multiplex reaction.

The invention further may preferably include a web based system foranalysis of the gene expression data. After running the preferredamplification array, e.g., PCR, a user will determine the regulatorystatus of a target sample and a control sample. In a preferredembodiment a user will effect comparison and analysis by the use of anavailable web based analysis tool or equivalent. In the context of thepresent invention this tool may in addition provide users with a number(index, probability or analogous parameter) which will indicate therelative regulatory status of a particular treatment sample compared toan appropriate control sample.

Applications of Present Invention

Diagnostic/Sample Classification Methods

The invention provides for methods of using the biomarker sets toanalyze a sample from an individual or subject so as to determine orclassify the subject's sample at a molecular level, to determine theregulation status of the IL-6/STATS pathway. The sample may or may notbe derived from a tumor. The individual need not actually be afflictedwith cancer, non-cancer inflammatory conditions, and/or any otherdisease. Essentially, the expression of specific biomarker genes in theindividual, or a sample taken therefrom, is compared to a standard orcontrol. For example, assume two cancer-related conditions, X and Y. Onecan compare the level of expression of IL-6/STAT3 pathway biomarkers forcondition X in an individual to the level of the biomarker-derivedpolynucleotides in a control, wherein the level represents the level ofexpression exhibited by samples having condition X. In this instance, ifthe expression of the markers in the individual's sample issubstantially (i.e., statistically) different from that of the control,then the individual does not have condition X. Where, as here, thechoice is bimodal (i.e., a sample is either X or Y), the individual canadditionally be said to have condition Y. Of course, the comparison to acontrol representing condition Y can also be performed. Preferably, bothare performed simultaneously, such that each control acts as both apositive and a negative control. The distinguishing result may thuseither be a demonstrable difference from the expression levels (i.e. theamount of marker-derived RNA, or polynucleotides derived therefrom)represented by the control, or no significant difference.

Thus, in one embodiment, the method of determining a particulartumor-related status of an individual comprises the steps of (1)hybridizing labeled target polynucleotides from an individual to amicroarray containing the above biomarker set or a subset of thebiomarkers; (2) hybridizing standard or control polynucleotide moleculesto the microarray, wherein the standard or control molecules aredifferentially labeled from the target molecules; and (3) determiningthe difference in transcript levels, or lack thereof, between the targetand standard or control, wherein the difference, or lack thereof,determines the individual's tumor-related status.

In a more specific embodiment, the standard or control moleculescomprise biomarker-derived polynucleotides from a pool of samples fromnormal individuals, a pool of samples from normal adjacent tissue, or apool of tumor samples from individuals with cancer. In a preferredembodiment, the standard or control is artificially-generated pool ofbiomarker-derived polynucleotides, which pool is designed to mimic thelevel of biomarker expression exhibited by clinical samples of normal orcancer tumor tissue having a particular clinical indication (e.g.,cancerous or non-cancerous; IL-6/STAT3 pathway regulated orderegulated). In another specific embodiment, the control moleculescomprise a pool derived from normal or cancer cell lines.

The present invention provides a set of biomarkers or a microarraycontaining useful for distinguishing the regulation status of theIL-6/STAT3 pathway, e.g., in a cell sample (tumor). Thus, in oneembodiment of the above method, the level of polynucleotides (i.e., mRNAor polynucleotides derived therefrom) in a sample from an individual,expressed from the 16 biomarkers provided herein are compared to thelevel of expression of the same biomarkers from a control. If thepurpose is to identify whether a compound affects IL-6/STAT3 signaling,then the control may comprise a sample treated by the same methodsexcept in the absence of the compound.

The comparison alternatively may be to both deregulated and regulatedIL-6/STAT3 signaling pathway tumor samples, and the comparison may be topolynucleotide pools from a number of deregulated and regulatedIL-6/STAT3 signaling pathway tumor samples, respectively. Where theindividual's biomarker expression most closely resembles or correlateswith the deregulated control, and does not resemble or correlate withthe regulated control, the individual is classified as having aderegulated IL-6/STAT3 signaling pathway. Where the pool is not purederegulated or regulated IL-6/STAT3 signaling pathway type tumorssamples, for example, a sporadic pool is used, a set of experimentsusing individuals with known IL-6/STAT3 signaling pathway status may behybridized against the pool in order to define the expression templatesfor the deregulated and regulated group. Each individual with unknownIL-6/STAT3 signaling pathway status is hybridized against the same pooland the expression profile is compared to the template(s) to determinethe individual's IL-6/STAT3 signaling pathway status. As noted in thepreferred methods the expression of the biomarkers is effected by use ofRT-PCR.

In another specific embodiment, the method comprises: (1) calculating ameasure of similarity between a first expression profile and aderegulated IL-6/STAT3 signaling pathway template, or calculating afirst measure of similarity between said first expression profile andsaid deregulated IL-6/STAT3 signaling pathway template and a secondmeasure of similarity between said first expression profile and aregulated IL-6/STAT3 signaling pathway template, said first expressionprofile comprising the expression levels of a first plurality of genesin the cell sample, said deregulated IL-6/STAT3 signaling pathwaytemplate comprising expression levels of said first plurality of genesthat are average expression levels of the respective genes in aplurality of cell samples having at least one or more components of saidIL-6/STAT3 signaling pathway with abnormal activity, and said regulatedIL-6/STAT3 signaling pathway template comprising expression levels ofsaid first plurality of genes that are average expression levels of therespective genes in a plurality of cell samples not having at least oneor more components of said IL-6/STAT3 signaling pathway with abnormalactivity, said first plurality of genes consisting of at least 5 of thegenes for which biomarkers are listed herein;

(2) classifying said cell sample as having said deregulated IL-6/STAT3signaling pathway if said first expression profile has a high similarityto said deregulated IL-6/STAT3 signaling pathway template or has ahigher similarity to said deregulated IL-6/STAT3 signaling pathwaytemplate than to said regulated IL-6/STAT3 signaling pathway template,or classifying said cell sample as having said regulated IL-6/STAT3signaling pathway if said first expression profile has a low similarityto said deregulated IL-6/STAT3 signaling pathway template or has ahigher similarity to said regulated IL-6/STAT3 signaling pathwaytemplate than to said deregulated IL-6/STAT3 signaling pathway template;wherein said first expression profile has a high similarity to saidderegulated IL-6/STAT3 signaling pathway template if the similarity tosaid deregulated IL-6/STAT3 signaling pathway template is above apredetermined threshold, or has a low similarity to said deregulatedIL-6/STAT3 signaling pathway template if the similarity to saidderegulated IL-6/STAT3 signaling pathway template is below saidpredetermined threshold; and

(3) displaying; or outputting to a user interface device, a computerreadable storage medium, or a local or remote computer system; theclassification produced by said classifying step (2).

In another specific embodiment, the set of biomarkers may be used toclassify a sample from a subject by the IL-6/STAT3 signaling pathwayregulation status. The sample may or may not be derived from a tumor.Thus, in one embodiment of the above method, the level ofpolynucleotides (i.e., mRNA or polynucleotides derived therefrom) in asample from an individual, expressed from the biomarkers provided hereinare compared to the level of expression of the same biomarkers from acontrol, wherein the control comprises biomarker-related polynucleotidesderived from deregulated IL-6/STAT3 signaling pathway samples, regulatedIL-6/STAT3 signaling pathway samples, or both. The comparison may be toboth deregulated and regulated IL-6/STAT3 signaling pathway samples, andthe comparison may be to polynucleotide pools from a number ofderegulated and regulated IL-6/STAT3 signaling pathway samples,respectively. The comparison may also be made to a mixed pool of sampleswith deregulated and regulated IL-6/STAT3 signaling pathway or unknownsamples.

For the above embodiments, the full set of biomarkers may be used (i.e.,the complete set of 16 biomarkers). In other embodiments, subsets of the16 biomarkers may be used, e.g., 1-15 of the 16 biomarkers, at least 5of the 16 biomarkers, at least 10-15 of the biomarkers.

In another embodiment, the expression profile is a differentialexpression profile comprising differential measurements of saidplurality of genes in a sample derived from a subject versusmeasurements of said plurality of genes in a control sample. Thedifferential measurements can be xdev, log(ratio), error-weightedlog(ratio), or a mean subtracted log(intensity) (see, e.g., PCTpublication WO00/39339, published on Jul. 6, 2000; PCT publicationWO2004/065545, published Aug. 5, 2004, each of which is incorporatedherein by reference in its entirety). The similarity between thebiomarker expression profile of a sample or an individual and that of acontrol can be assessed a number of ways using any method known in theart. For example, Dai et al. describe a number of different ways ofcalculating gene expression templates and corresponding biomarker genetsuseful in classifying breast cancer patients (U.S. Pat. No. 7,171,311;WO2002/103320; WO2005/086891; WO2006015312; WO2006/084272). Similarly,Linsley et al. (US2003/0104426) and Radish et al. (US20070154931)disclose gene biomarker genesets and methods of calculating geneexpression templates useful in classifying chronic myelogenous leukemiapatients. In the simplest case, the profiles can be compared visually ina printout of expression difference data. Alternatively, the similaritycan be calculated mathematically.

In one embodiment, the similarity is represented by a correlationcoefficient between the patient or sample profile and the template. Inone embodiment, a correlation coefficient above a correlation thresholdindicates high similarity, whereas a correlation coefficient below thethreshold indicates low similarity. In some embodiments, the correlationthreshold is set as 0.3, 0.4, 0.5, or 0.6. In another embodiment,similarity between a sample or patient profile and a template isrepresented by a distance between the sample profile and the template.In one embodiment, a distance below a given value indicates a highsimilarity, whereas a distance equal to or greater than the given valueindicates low similarity.

Thus, in a more specific embodiment, the above method of determining aparticular tumor-related status of an individual comprises the steps of(1) hybridizing labeled target polynucleotides from an individual to amicroarray containing one of the above marker sets; (2) hybridizingstandard or control polynucleotides molecules to the microarray, whereinthe standard or control molecules are differentially labeled from thetarget molecules; and (3) determining the ratio (or difference) oftranscript levels between two channels (individual and control), orsimply the transcript levels of the individual; and (4) comparing theresults from (3) to the predefined templates, wherein said determiningis accomplished by any means known in the art, and wherein thedifference, or lack thereof, determines the individual's tumor-relatedstatus. The method can use the complete set of 16 biomarkers. However,subsets of the 16 biomarkers may also be used (e.g., at least 5 of the16 biomarkers, at least 10-15 of the biomarkers).

In yet another embodiment, the signature score of a sample is defined asthe average expression level (such as mean log(ratio)) of the completeset of 16 biomarkers or a subset of these biomarkers. If the signaturescore for a sample is above a pre-determined threshold, then the sampleis considered to have deregulation of the IL-6/STATS signaling pathway.The pre-determined threshold may be 0, or may be the mean, median, or apercentile of signature scores of a collection of samples or a pooledsample used as a standard or control.

The use of the biomarkers is not limited to distinguishing orclassifying particular tumor types, such as liver cancer, as havingderegulated or regulated IL-6/STAT3 signaling pathway. The biomarkersmay be used to classify cell samples from any cancer type, whereaberrant IL-6/STAT3 signaling may be implicated such as lung, breast,esophageal, head and neck, colonic, gastrointestinal, prostate, multiplemyeloma, hepatic, ovarian, neuroblastoma, glioblastoma, melanoma,pancreatic adenocarcinoma, renal cell carcinoma, cholangiocellularcarcinoma, and various leukemias and lymphomas. Non-limiting examplesthereof include low grade/follicular non-Hodgkin's lymphoma (NHL), smalllymphocytic (SL) NHL, intermediate grade/follicular NHL, intermediategrade diffuse NHL, chronic lymphocytic leukemia (CLL), high gradeimmunoblastic NHL, high grade lymphoblastic NHL, high grade smallnoncleaved cell NHL, bulky disease NHL, mantle cell lymphoma,AIDS-related lymphoma, Waldenstrom's Macroglobulinemia and T celllymphomas and leukemias

The use of the biomarkers is also not restricted to distinguishing orclassifying cell samples as having deregulated or regulated IL-6/STAT3signaling pathway for cancer-related conditions, and may be applied in avariety of phenotypes or conditions, in which aberrant IL-6/STAT3signaling plays a role, or the level of IL-6/STAT3 signaling activity issought. For example, the biomarkers may be useful for classifyingsamples for non-cancer inflammatory conditions, including, but notlimited to, hypoferremia of inflammation, acute-phase response toinflammation and infection, chronic inflammation, inflammation incardiovascular, systemic juvenile rheumatoid arthritis, Staphylococcusepidermidis-induced peritoneal inflammation, and pulmonary inflammation,e.g., adult respiratory distress syndrome, shock lung, chronic pulmonaryinflammatory disease, pulmonary sarcoidosis, pulmonary fibrosis andsilicosis. The IL-6/STAT3 signaling pathway has previously beenimplicated in the inflammatory response (Grivennikov S I, Karin M.Dangerous liaisons: STAT3 and NF-kappaB collaboration and crosstalk incancer. Cytokine Growth Factor Rev. 2010 February; 21(1):11-9. Epub 2009Dec. 16); biological events such as, e.g., embryonic development,programmed cell death, organogenesis, innate immunity, adaptive immunityand cell growth regulation in many organisms (Mankan A K, Greten F R.Inhibiting signal transducer and activator of transcription 3:rationality and rationale design of inhibitors. Expert Opin InvestigDrugs. 2011 September; 20(9):1263-75. Epub 2011 Jul. 14); and cancerinitiation and progression (Yu H, Pardoll D, Jove R. STATs in cancerinflammation and immunity: a leading role for STAT3. Nat Rev Cancer.2009 November; 9(11):798-809).

Methods of Predicting Response to Treatment and Assigning Treatment

The invention provides a set of biomarkers useful for distinguishingsamples from those patients likely to respond to treatment with an agentthat modulates the IL-6/STAT3 signaling pathway, from patients who arenot likely to respond to treatment an agent that modulates theIL-6/STAT3 signaling pathway. Thus, the invention further provides amethod for using these biomarkers for determining whether an individualwith cancer is a predicted responder to treatment with an agent thatmodulates the IL-6/STAT3 signaling pathway. In one embodiment, theinvention provides for a method of predicting response of a cancerpatient to an agent that modulates the IL-6/STAT3 signaling pathwaycomprising (1) comparing the level of expression of the 16 biomarkers ina sample taken from the individual to the level of expression of thesame biomarkers in a standard or control, where the standard or controllevels represent those found in a sample having a deregulated IL-6/STAT3signaling; and (2) determining whether the level of thebiomarker-related polynucleotides in the sample from the individual issignificantly different than that of the control, wherein if nosubstantial difference is found, the patient is predicted to respond totreatment with an agent that modulates the IL-6/STAT3 signaling pathway,and if a substantial difference is found, the patient is predicted notto respond to treatment with an agent that modulates the IL-6/STAT3signaling pathway. Persons of skill in the art will readily see that thestandard or control levels may be from a sample having a regulatedIL-6/STAT3 signaling pathway. In a more specific embodiment, bothcontrols are run. In case the pool is not pure “IL-6/STAT3 regulated” or“IL-6/STAT3 deregulated,” a set of experiments of individuals with knownresponder status may be hybridized against the pool to define theexpression templates for the predicted responder and predictednon-responder group. Each individual with unknown outcome is hybridizedagainst the same pool and the resulting expression profile is comparedto the templates to predict its outcome.

IL-6/STAT3 signaling pathway deregulation status of a tumor may indicatea subject that is responsive to treatment with an agent that modulatesthe IL-6/STAT3 signaling pathway. Therefore, the invention provides fora method of determining or assigning a course of treatment of a cancerpatient, comprising determining whether the level of expression of the16 biomarkers, or a subset thereof, correlates with the level of thesebiomarkers in a sample representing deregulated IL-6/STAT3 signalingpathway status or regulated IL-6/STAT3 signaling pathway status; anddetermining or assigning a course of treatment, wherein if theexpression correlates with the deregulated IL-6/STAT3 signaling pathwaystatus pattern, the tumor is treated with an agent that modulates theIL-6/STAT3 signaling pathway.

As with the diagnostic biomarkers, the method can preferably use thecomplete set of 16 biomarkers. However, subsets of the 16 biomarkers mayalso be used (e.g., at least 5 of the 16 biomarkers, at least 10-15 ofthe biomarkers).

Classification of a sample as “predicted responder” or “predictednon-responder” is accomplished substantially as for the diagnosticbiomarkers described above, wherein a template is generated to which thebiomarker expression levels in the sample are compared.

In another embodiment, the above method for measuring the effect of anagent on the IL-6/STATS signaling pathway is preferably determined afterreal-time PCR measuring expression levels of 16 biomarker genes usingSYBR Green, and a ΔΔCT method employed to analysis the data. The averageCT values of house keeping genes in each sample, e.g., 8 housekeepinggenes, is calculated as house keeping gene CT value for that sample. ΔCTwas calculated by subtracting house keeping CT value from individualassay CT value of same sample. ΔΔCT value was derived by furthersubtracting ΔCT value of control samples of each assay from itscorresponding ΔCT value of treatment sample. This ΔΔCT value of those 16genes is then compared to ΔΔCT value of 16 genes in a training data poolthat contains several samples, e.g., 16 total sample, 7 of which arenegatively regulated and 9 of which are positively regulated in terms ofpathway activity. A random forest method is preferably used to analyzethe ΔΔCT values of the samples and the expression thereof used to assessthe regulatory status of the IL-6/STAT3 pathway activity in the sample.

The use of the biomarkers is not restricted to predicting response toagents that modulate IL-6/STAT3 signaling pathway for cancer-relatedconditions, and may be applied in a variety of phenotypes or conditions,clinical or experimental, in which gene expression plays a role. Where aset of biomarkers has been identified that corresponds to two or morephenotypes, the biomarker sets can be used to distinguish thesephenotypes. For example, the phenotypes may be the diagnosis and/orprognosis of clinical states or phenotypes associated with cancers andother disease conditions, or other physiological conditions, predictionof response to agents that modulate pathways other than the IL-6/STAT3signaling pathway, wherein the expression level data is derived from aset of genes correlated with the particular physiological or diseasecondition.

The use of the biomarkers is not limited to predicting response toagents that modulate IL-6/STAT3 signaling pathway for a particularcancer type, such as liver cancer. The biomarkers may be used to predictresponse to agents in any cancer type where aberrant IL-6/STAT3signaling may be implicated. Aberrant IL-6/STAT3 pathway signaling hasbeen discovered in a wide variety of cancers, including lung, breast,esophageal, head and neck, colonic, gastrointestinal, prostate, multiplemyeloma, hepatic, ovarian, neuroblastoma, glioblastoma, melanoma,pancreatic adenocarcinoma, renal cell carcinoma, cholangiocellularcarcinoma, and various leukemias and lymphomas.

The use of the biomarkers is also not restricted to predicting responseto agents that modulate IL-6/STAT3 signaling pathway for cancer-relatedconditions, and may be applied in a variety of phenotypes or conditions,in which aberrant IL-6/STAT3 signaling plays a role, or the level ofIL-6/STAT3 signaling activity is sought. For example, the biomarkers maybe useful for classifying samples for non-cancer inflammatoryconditions, such as, but not limited to, hypoferremia of inflammation,acute-phase response to inflammation and infection, chronicinflammation, inflammation in cardiovascular, systemic juvenilerheumatoid arthritis, Staphylococcus epidermidis-induced peritonealinflammation, and pulmonary inflammation, e.g., adult respiratorydistress syndrome, shock lung, chronic pulmonary inflammatory disease,pulmonary sarcoidosis, pulmonary fibrosis and silicosis. The IL-6/STAT3signaling pathway has previously been implicated in the inflammatoryresponse (Grivennikov S I, Karin M. Dangerous liaisons: STAT3 andNF-kappaB collaboration and crosstalk in cancer. Cytokine Growth FactorRev. 2010 February; 21(1):11-9. Epub 2009 Dec. 16); biological eventssuch as, e.g., embryonic development, programmed cell death,organogenesis, innate immunity, adaptive immunity and cell growthregulation in many organisms (Mankan A K, Greten F R. Inhibiting signaltransducer and activator of transcription 3: rationality and rationaledesign of inhibitors. Expert Opin Investig Drugs. 2011 September;20(9):1263-75. Epub 2011 Jul. 14); and cancer initiation and progression(Yu H, Pardo11 D, Jove R. STATs in cancer inflammation and immunity: aleading role for STAT3. Nat Rev Cancer. 2009 November; 9(11):798-809).Additionally, the biomarkers can be used to detect inflammation sites invivo or ex vivo.

Method of Determining Whether an Agent Modulates the IL-6/STAT3Signaling Pathway

The invention provides a set of biomarkers useful for and methods ofusing the biomarkers for identifying or evaluating an agent that ispredicted to modify or modulate the IL-6/STAT3 signaling pathway in asubject. “IL-6/STAT3 signaling pathway” is initiated by the cytokineIL-6 binding to the IL-6 receptor (IL-6R), and this engagement of IL6 toits specific receptor activates receptor-associated tyrosine kinaseJanus Kinase 2 (JAK2), which in turn phosphorylates tyrosine residues inthe cytoplasmic tail of the IL-6R that function as docking sites forSTAT3. JAK2-dependent phosphorylation of STAT3 leads to itshomodimerization and nuclear translocation. Once in the nucleus,activated STAT3 functions as transcriptional activator, inducingexpression of target genes (Levy D E, Darnell JE Jr. Stats:transcriptional control and biological impact. Nat Rev Mal Cell Biol.2002 September; 3(9):651-62). STAT3 induces expression of a large numberof genes having a STAT3 binding site, including genes involved in cellsurvivial, cell proliferation, invasion, angiogenesis, and tumor immuneevasion (e.g., cyclin DI, p53, Bcl-Xl, MMP-2, MMP-9, VEGF, bFGF, HIF-1alpha, IP-10, and RANTES) and feedback regulation of the pathway (e.g.,SOCS3).

Agents affecting the IL-6/STAT3 signaling pathway include small moleculecompounds; proteins or peptides (including antibodies); siRNA, shRNA, ormicroRNA molecules; or any other agents that modulate one or more genesor proteins that function within the IL-6/STAT3 signaling pathway orother signaling pathways that interact with the IL-6/STAT3 signalingpathway, such as the Notch pathway.

“IL-6/STAT3 pathway agent” refers to an agent which modulates theIL-6/STAT3 pathway signaling. A IL-6/STAT3 pathway inhibitor inhibitsthe IL-6/STAT3 pathway signaling. Molecular targets of such agents mayinclude JAK2 and STAT3, as well as any of the genes listed herein. Suchagents are known in the art and include, but are not limited to: AZD1480(Hedvat et al., The JAK2 Inhibitor, AZD1480, Potently Blocks Stat3Signaling and Oncogenesis in Solid Tumors. Cancer Cell. 2009 Dec. 8;16(6):487-97), WP1066 (Calbiochem, La Jolla, Calif., USA), NSC 74859,Stattic (Santa Cruz Biotechnology, Inc.; Schust et al., Stattic: A SmallMolecule Inhibitor of STAT3 Activation and Dimerization. Chemistry &Biology. 2006; 13:1235-1242), and LLL12 (Liu et al., Inhibition of STAT3signaling blocks the anti-apoptotic activity of IL-6 in human livercancer cells. J Biol Chem, 285:27429-39, Epub 2010 Jun. 18).

In one embodiment, the method for measuring the effect or determiningwhether an agent modulates the IL-6/STAT3 signaling pathway comprises:(1) comparing the level of expression of the 16 biomarkers in a sampletreated with an agent to the level of expression of the same biomarkersin a standard or control, wherein the standard or control levelsrepresent those found in a vehicle-treated sample; and (2) determiningwhether the level of the biomarker-related polynucleotides in thetreated sample is significantly different than that of thevehicle-treated control, wherein if no substantial difference is found,the agent is predicted not to have an modulate the IL-6/STAT3 signalingpathway, and if a substantial difference is found, the agent ispredicted to modulate the IL-6/STAT3 signaling pathway.

In another embodiment, the above method for measuring the effect of anagent on the TL-6/STAT3 signaling pathway is preferably determined afterreal-time PCR measuring expression levels of 16 biomarker genes (e.g.,using SYBR green), and a ΔΔCT method employed to analysis the data. Theaverage CT values of house keeping genes in each sample is calculated ashouse keeping gene CT value for that sample. ΔCT was calculated bysubtracting house keeping CT value from individual assay CT value ofsame sample. ΔΔCT value was derived by further subtracting ΔCT value ofcontrol samples of each assay from its corresponding ΔCT value oftreatment sample. This ΔΔCt value of those 16 genes is then compared toΔΔCt value of 16 genes in a training data pool that contains severalsamples (e.g., 16 total sample, 7 negatively regulated and 9 positivelyregulated in terms of pathway activity). A random forest method ispreferably used to analyze the ΔΔCT values of the samples and theexpression thereof used to assess the regulatory status of theIL-6/STAT3 pathway activity in the sample.

The use of the biomarkers is not restricted to determining whether anagent modulates IL-6/STAT3 signaling pathway for cancer-relatedconditions, and may be applied in a variety of phenotypes or conditions,clinical or experimental, in which gene expression plays a role. Where aset of biomarkers has been identified that corresponds to two or morephenotypes, the biomarker sets can be used to distinguish thesephenotypes. For example, the phenotypes may be the diagnosis and/orprognosis of clinical states or phenotypes associated with cancers andother disease conditions, or other physiological conditions, predictionof response to agents that modulate pathways other than the IL-6/STAT3signaling pathway, wherein the expression level data is derived from aset of genes correlated with the particular physiological or diseasecondition.

The use of the biomarkers is not limited to determining whether an agentmodulates the IL-6/STAT3 signaling pathway for a particular cancer type,such as liver cancer. The biomarkers may be used to determine whether anagent modulates the IL-6/STAT3 for any cancer type, where aberrantIL-6/STAT3 signaling may be implicated. Aberrant IL-6/STAT3 pathwaysignaling has been discovered in a wide variety of cancers, includinglung, breast, esophageal, head and neck, colonic, gastrointestinal,prostate, multiple myeloma, hepatic, ovarian, neuroblastoma,glioblastoma, melanoma, pancreatic adenocarcinoma, renal cell carcinoma,cholangiocellular carcinoma, and various leukemias, and lymphomas.

The use of the biomarkers is also not restricted determining whether anagent modulates the IL-6/STAT3 signaling pathway for cancer-relatedconditions, and may be applied for agents for a variety of phenotypes orconditions, in which aberrant IL-6/STAT3 signaling plays a role, or thelevel of IL-6/STAT3 signaling activity is sought. For example, thebiomarkers may be useful for classifying samples for non-cancerinflammatory conditions, such as, but not limited to, hypoferremia ofinflammation, acute-phase response to inflammation and infection,chronic inflammation, inflammation in cardiovascular, systemic juvenilerheumatoid arthritis, Staphylococcus epidermidis-induced peritonealinflammation, and pulmonary inflammation, e.g., adult respiratorydistress syndrome, shock lung, chronic pulmonary inflammatory disease,pulmonary sarcoidosis, pulmonary fibrosis and silicosis. The IL-6/STAT3signaling pathway has previously been implicated in the inflammatoryresponse (Grivennikov S I, Karin M. Dangerous liaisons: STAT3 andNF-kappaB collaboration and crosstalk in cancer. Cytokine Growth FactorRev. 2010 February; 21(1):11-9. Epub 2009 Dec. 16); biological eventssuch as, e.g., embryonic development, programmed cell death,organogenesis, innate immunity, adaptive immunity and cell growthregulation in many organisms (Mankan A K, Greten F R. Inhibiting signaltransducer and activator of transcription 3: rationality and rationaledesign of inhibitors. Expert Opin Investig Drugs. 2011 September;20(9):1263-75. Epub 2011 Jul. 14); and cancer initiation and progression(Yu H, Pardoll D, Jove R. STATs in cancer inflammation and immunity: aleading role for STAT3. Nat Rev Cancer. 2009 November; 9(11):798-809).Additionally, the biomarkers can be used to detect inflammation sites invivo or ex vivo.

Method of Measuring Phartnacodynamic Effect of an Agent

The invention provides a set of biomarkers useful for measuring thepharmacodynamic effect of an agent on the IL-6/STAT3 signaling pathway.The biomarkers provided may be used to monitor modulation of theIL-6/STAT3 signaling pathway at various time points following treatmentwith said agent in a patient or sample. Thus, the invention furtherprovides a method for using these biomarkers as an early evaluation forefficacy of an agent which modulates the IL-6/STAT3 signaling pathway.In one embodiment, the invention provides for a method of measuringpharmacodynamic effect of an agent that modulates the IL-6/STAT3signaling pathway in patient or sample comprising: (1) comparing thelevel of expression of the 16 biomarkers in a sample treated with anagent to the level of expression of the same biomarkers in a standard orcontrol, wherein the standard or control levels represent those found ina vehicle-treated sample; and (2) determining whether the level of thehiomarker-related polynucleotides in the treated sample is significantlydifferent than that of the vehicle-treated control, wherein if nosubstantial difference is found, the agent is predicted not to have anpharmacodynamic effect on the IL-6/STAT3 signaling pathway, and if asubstantial difference is found, the agent is predicted to have anpharmacodynamic effect on the IL-6/STAT3 signaling pathway. In anotherspecific embodiment, the invention provides a subset of at least 5biomarkers, or at least 10 biomarkers, drawn from the set of 16 that canbe used to monitor pharmacodynamic activity of an agent on theIL-6/STAT3 signaling pathway.

In another embodiment, the above method for measuring the effect of anagent on the IL-6/STAT3 signaling pathway is preferably determined afterreal-time PCR measuring expression levels of 16 biomarker genes (e.g.,using SYBR green detection), and a ΔΔCT method employed to analysis thedata. The average CT values of house keeping genes in each sample iscalculated as house keeping gene CT value for that sample. ΔCT wascalculated by subtracting house keeping CT value from individual assayCT value of same sample. ΔΔCT value was derived by further subtractingΔCT value of control samples of each assay from its corresponding ΔCTvalue of treatment sample. This ΔΔCt value of those 16 genes is thencompared to ΔΔCt value of 16 genes in a training data pool that containsseveral samples (e.g., 16 total sample, 7 negatively regulated and 9positively regulated in terms of pathway activity). A random forestmethod is preferably used to analyze the ΔΔCT values of the samples andthe expression thereof used to assess the regulatory status of theIL-6/STAT3 pathway activity in the sample.

Improving Sensitivity to Expression Level Differences

In using the biomarkers disclosed herein, and, indeed, using any sets ofbiomarkers to differentiate an individual or subject having onephenotype from another individual or subject having a second phenotype,one can compare the absolute expression of each of the biomarkers in asample to a control; for example, the control can be the average levelof expression of each of the biomarkers, respectively, in a pool ofindividuals or subjects. To increase the sensitivity of the comparison,however, the expression level values are preferably transformed in anumber of ways.

For example, the expression level of each of the biomarkers can benormalized by the average expression level of all markers the expressionlevel of which is determined, or by the average expression level of aset of control genes. Thus, in one embodiment, the biomarkers arerepresented by probes on a microarray, and the expression level of eachof the biomarkers is normalized by the mean or median expression levelacross all of the genes represented on the microarray, including anynon-biomarker genes. In a specific embodiment, the normalization iscarried out by dividing the median or mean level of expression of all ofthe genes on the microarray. In another embodiment, the expressionlevels of the biomarkers is normalized by the mean or median level ofexpression of a set of control biomarkers. In a specific embodiment, thecontrol biomarkers comprise a set of housekeeping genes. In anotherspecific embodiment, the normalization is accomplished by dividing bythe median or mean expression level of the control genes.

The sensitivity of a biomarker-based assay will also be increased if theexpression levels of individual biomarkers are compared to theexpression of the same biomarkers in a pool of samples. Preferably, thecomparison is to the mean or median expression level of each thebiomarker genes in the pool of samples. Such a comparison may beaccomplished, for example, by dividing by the mean or median expressionlevel of the pool for each of the biomarkers from the expression leveleach of the biomarkers in the sample. This has the effect ofaccentuating the relative differences in expression between biomarkersin the sample and markers in the pool as a whole, making comparisonsmore sensitive and more likely to produce meaningful results that theuse of absolute expression levels alone. The expression level data maybe transformed in any convenient way: preferably, the expression leveldata for all is log transformed before means or medians are taken.

In performing comparisons to a pool, two approaches may be used. First,the expression levels of the markers in the sample may be compared tothe expression level of those markers in the pool, where nucleic acidderived from the sample and nucleic acid derived from the pool arehybridized during the course of a single experiment. Such an approachrequires that new pool nucleic acid be generated for each comparison orlimited numbers of comparisons, and is therefore limited by the amountof nucleic acid available. Alternatively, and preferably, the expressionlevels in a pool, whether normalized and/or transformed or not, arestored on a computer, or on computer-readable media, to be used incomparisons to the individual expression level data from the sample(i.e., single-channel data).

Thus, the current invention provides the following method of classifyinga first cell or organism as having one of at least two differentphenotypes, where the different phenotypes comprise a first phenotypeand a second phenotype. The level of expression of each of a pluralityof genes in a first sample from the first cell or organism is comparedto the level of expression of each of said genes, respectively, in apooled sample from a plurality of cells or organisms, the plurality ofcells or organisms comprising different cells or organisms exhibitingsaid at least two different phenotypes, respectively, to produce a firstcompared value. The first compared value is then compared to a secondcompared value, wherein said second compared value is the product of amethod comprising comparing the level of expression of each of saidgenes in a sample from a cell or organism characterized as having saidfirst phenotype to the level of expression of each of said genes,respectively, in the pooled sample. The first compared value is thencompared to a third compared value, wherein said third compared value isthe product of a method comprising comparing the level of expression ofeach of the genes in a sample from a cell or organism characterized ashaving the second phenotype to the level of expression of each of thegenes, respectively, in the pooled sample. Optionally, the firstcompared value can be compared to additional compared values,respectively, where each additional compared value is the product of amethod comprising comparing the level of expression of each of saidgenes in a sample from a cell or organism characterized as having aphenotype different from said first and second phenotypes out includedamong the at least two different phenotypes, to the ievei of expressionof each of said genes, respectively, in said pooled sample. Finally, adetermination is made as to which of said second, third, and, ifpresent, one or more additional compared values, said first comparedvalue is most similar, wherein the first cell or organism is determinedto have the phenotype of the cell or organism used to produce saidcompared value most similar to said first compared value.

In a specific embodiment of this method, the compared values are eachratios of the levels of expression of each of said genes. In anotherspecific embodiment, each of the levels of expression of each of thegenes in the pooled sample is normalized prior to any of the comparingsteps. In a more specific embodiment, the normalization of the levels ofexpression is carried out by dividing by the median or mean level of theexpression of each of the genes or dividing by the mean or median levelof expression of one or more housekeeping genes in the pooled samplefrom said cell or organism. In another specific embodiment, thenormalized levels of expression are subjected to a log transform, andthe comparing steps comprise subtracting the log transform from the logof the levels of expression of each of the genes in the sample. Inanother specific embodiment, the two or more different phenotypes aredifferent regulation status of the IL-6/STAT3 signaling pathway. Instill another specific embodiment, the two or more different phenotypesare different predicted responses to treatment with an agent thatmodulates the IL-6/STAT3 signaling pathway. In yet another specificembodiment, the levels of expression of each of the genes, respectively,in the pooled sample or said levels of expression of each of said genesin a sample from the cell or organism characterized as having the firstphenotype, second phenotype, or said phenotype different from said firstand second phenotypes, respectively, are stored on a computer or on acomputer-readable medium.

In another specific embodiment, the two phenotypes are deregulated orIL-6/STAT3 signaling pathway status. In another specific embodiment, thetwo phenotypes are predicted IL-6/STAT3 signaling pathway-agentresponder status. In yet another specific embodiment, the two phenotypesare pharmacodynamic effect and no pharmacodynamic effect of an agent onthe IL-6/STAT3 signaling pathway.

In another specific embodiment, the comparison is made between theexpression of each of the genes in the sample and the expression of thesame genes in a pool representing only one of two or more phenotypes. Inthe context of IL-6/STAT3 signaling pathway status-correlated genes, forexample, one can compare the expression levels of IL-6/STAT3 signalingpathway regulation status-related genes in a sample to the average levelof the expression of the same genes in a “deregulated” pool of samples(as opposed to a pool of samples that include samples from patientshaving regulated and deregulated IL-6/STAT3 signaling pathway status).Thus, in this method, a sample is classified as having a deregulatedIL-6/STAT3 signaling pathway status if the level of expression ofprognosis-correlated genes exceeds a chosen coefficient of correlationto the average “deregulated IL-6/STAT3 signaling pathway” expressionprofile (i.e., the level of expression of IL-6/STAT3 signaling pathwaystatus-correlated genes in a pool of samples from patients having a“deregulated IL-6/STAT3 signaling pathway status.” Patients or subjectswhose expression levels correlate more poorly with the “deregulatedIL-6/STAT3 signaling pathway” expression profile (i.e., whosecorrelation coefficient fails to exceed the chosen coefficient) areclassified as having a regulated IL-6/STAT3 signaling pathway status.

Of course, single-channel data may also be used without specificcomparison to a mathematical sample pool. For example, a sample may beclassified as having a first or a second phenotype, wherein the firstand second phenotypes are related, by calculating the similarity betweenthe expression of at least 5 markers in the sample, where the markersare correlated with the first or second phenotype, to the expression ofthe same markers in a first phenotype template and a second phenotypetemplate, by (a) labeling nucleic acids derived from a sample with afluorophore to obtain a pool of fluorophore-labeled nucleic acids; (b)contacting said fluorophore-labeled nucleic acid with a microarray underconditions such that hybridization can occur, detecting at each of aplurality of discrete loci on the microarray a fluorescent emissionsignal from said fluorophore-labeled nucleic acid that is bound to saidmicroarray under said conditions; and (c) determining the similarity ofmarker gene expression in the individual sample to the first and secondtemplates, wherein if said expression is more similar to the firsttemplate, the sample is classified as having the first phenotype, and ifsaid expression is more similar to the second template, the sample isclassified as having the second phenotype.

Methods for Classification of Expression Profiles

In preferred embodiments, the methods of the invention use a classifierfor predicting IL-6/STAT3 signaling pathway regulation status of asample, predicting response to agents that modulate the IL-6/STAT3signaling pathway, assigning treatment to a subject, and/or measuringpharmacodynamic effect of an agent. The classifier can be based on anyappropriate pattern recognition method that receives an input comprisinga biomarker profile and provides an output comprising data indicatingwhich patient subset the patient belongs. The classifier can be trainedwith training data from a training population of subjects. Typically,the training data comprise for each of the subjects in the trainingpopulation a training marker profile comprising measurements ofrespective gene products of a plurality of genes in a suitable sampletaken from the patient and outcome information, i.e., deregulated orregulated IL-6/STAT3 signaling pathway status.

In preferred embodiments, the classifier can be based on aclassification (pattern recognition) method described below, e.g.,profile similarity; artificial neural network; support vector machine(SVM); logic regression, linear or quadratic discriminant analysis,decision trees, clustering, principal component analysis, nearestneighbor classifier analysis, nearest shrunken centroid, random forest.Such classifiers can be trained with the training population usingmethods described in the relevant sections, infra.

The biomarker profile can be obtained by measuring the plurality of geneproducts in a cell sample from the subject using a method known in theart, e.g., a method described infra.

Various known statistical pattern recognition methods can be used inconjunction with the present invention. A classifier based on any ofsuch methods can be constructed using the biomarker profiles andIL-6/STAT3 pathway signaling status data of training patients. Such aclassifier can then be used to evaluate the IL-6/STAT3 pathway signalingstatus of a patient based on the patient's biomarker profile. Themethods can also be used to identify biomarkers that discriminatebetween different IL-6/STAT3 signaling pathway regulation status using abiomarker profile and IL-6/STAT3 signaling pathway regulation data oftraining patients.

Profile Matching

A subject can be classified by comparing a biomarker profile obtained ina suitable sample from the subject with a biomarker profile that isrepresentative of a particular phenotypic state. Such a marker profileis also termed a “template profile” or a “template.” The degree ofsimilarity to such a template profile provides an evaluation of thesubject's phenotype. If the degree of similarity of the subject markerprofile and a template profile is above a predetermined threshold, thesubject is assigned the classification represented by the template. Forexample, a subject's outcome prediction can be evaluated by comparing abiomarker profile of the subject to a predetermined template profilecorresponding to a given phenotype or outcome, e.g., a IL-6/STAT3signaling pathway template comprising measurements of the plurality ofbiomarkers which are representative of levels of the biomarkers in aplurality of subjects that have tumors with deregulated IL-6/STAT3signaling pathway status.

In one embodiment, the similarity is represented by a correlationcoefficient between the subject's profile and the template. In oneembodiment, a correlation coefficient above a correlation thresholdindicates a high similarity, whereas a correlation coefficient below thethreshold indicates a low similarity.

Artificial Neural Network

In some embodiments, a neural network is used. A neural network can beconstructed for a selected set of molecular markers of the invention. Aneural network is a two-stage regression or classification model. Aneural network has a layered structure that includes a layer of inputunits (and the bias) connected by a layer of weights to a layer ofoutput units. For regression, the layer of output units typicallyincludes just one output unit. However, neural networks can handlemultiple quantitative responses in a seamless fashion. In multilayerneural networks, there are input units (input layer), hidden units(hidden layer), and output units (output layer). There is, furthermore,a single bias unit that is connected to each unit other than the inputunits. Neural networks are described in Duda et al., 2001, PatternClassification, Second Edition, John Wiley & Sons, Inc., New York; andHastie et al., 2001, The Elements of Statistical Learning,Springer-Verlag, New York.

Support Vector Machine

In some embodiments of the present invention, support vector machines(SVMs) are used to classify subjects using expression profiles of markergenes described in the present invention. General description of SVM canbe found in, for example, Cristianini and Shawe-Taylor, 2000, AnIntroduction to Support Vector Machines, Cambridge University Press,Cambridge, Baser et al., 1992, “A training algorithm for optimal marginclassifiers, in Proceedings of the 5th Annual ACM Workshop onComputational Learning Theory, ACM Press, Pittsburgh, Pa., pp. 142-152;Vapnik, 1998, Statistical Learning Theory, Wiley, New York; Duda,Pattern Classification, Second Edition, 2001, John Wiley & Sons, Inc.;Hastie, 2001, The Elements of Statistical Learning, Springer, N.Y.; andFurey et al, 2000, Bioinformatics 16, 906-914. Applications of SVM inbiological applications are described in Jaakkola et al., Proceedings ofthe 7th International Conference on Intelligent Systems for MolecularBiology, AAAI Press, Menlo Park, Calif. (1999); Brown et al., Proc.Natl. Acad. Sci. 97(1):262-67 (2000); Zien et al., Bioinformatics,16(9):799-807 (2000); Furey et al., Bioinformatics, 16(10):906-914(2000).

In some embodiments, the classifier is based on a regression model,preferably a logistic regression model. Such a regression model includesa coefficient for each of the molecular markers in a selected set ofmolecular biomarkers of the invention. In such embodiments, thecoefficients for the regression model are computed using, for example, amaximum likelihood approach. In particular embodiments, molecularbiomarker data from two different classification or phenotype groups,e.g., deregulated or regulated IL-6/STATS signaling pathway, response ornon-response to treatment to an agent that modulates the IL-6/STAT3signaling pathway, is used and the dependent variable is the phenotypicstatus of the patient for which molecular marker characteristic data arefrom.

Some embodiments of the present invention provide generalizations of thelogistic regression model that handle multicategory (polychotomous)responses. Such embodiments can be used to discriminate an organism intoone or three or more classification groups, e.g., good, intermediate,and poor therapeutic response to treatment with IL-6/STAT3 signalingpathway agents. Such regression models use multicategory logic modelsthat simultaneously refer to all pairs of categories, and describe theodds of response in one category instead of another. Once the modelspecifies logits for a certain (J-1) pairs of categories, the rest areredundant. See, for example, Agresti, An Introduction to CategoricalData Analysis, John Wiley & Sons, Inc., 1996, New York, Chapter 8, whichis hereby incorporated by reference.

Discriminant Analysis

Linear discriminant analysis (LDA) attempts to classify a subject intoone of two categories based on certain object properties. In otherwords, LDA tests whether object attributes measured in an experimentpredict categorization of the objects. LDA typically requires continuousindependent variables and a dichotomous categorical dependent variable.In the present invention, the expression values for the selected set ofmolecular markers of the invention across a subset of the trainingpopulation serve as the requisite continuous independent variables. Theclinical group classification of each of the members of the trainingpopulation serves as the dichotomous categorical dependent variable.

LDA seeks the linear combination of variables that maximizes the ratioof between-group variance and within-group variance by using thegrouping information. Implicitly, the linear weights used by LDA dependon how the expression of a molecular biomarker across the training setseparates in the two groups (e.g., a group that has deregulatedIL-6/STAT3 signaling pathway and a group that have regulated IL-6/STAT3signaling pathway status) and how this gene expression correlates withthe expression of other genes. In some embodiments, LDA is applied tothe data matrix of the N members in the training sample by K genes in acombination of genes described in the present invention. Then, thelinear discriminant of each member of the training population isplotted. Ideally, those members of the training population representinga first subgroup (e.g. those subjects that have deregulated IL-6/STAT3signaling pathway status) will cluster into one range of lineardiscriminant values (e.g., negative) and those member of the trainingpopulation representing a second subgroup (e.g. those subjects that haveregulated IL-6/STAT3 signaling pathway status) will cluster into asecond range of linear discriminant values (e.g., positive). The LDA isconsidered more successful when the separation between the clusters ofdiscriminant values is larger. For more information on lineardiscriminant analysis, see Duda, Pattern Classification, Second Edition,2001, John Wiley & Sons, Inc; and Hastie, 2001, The Elements ofStatistical Learning, Springer, N.Y.; Venables & Ripley, 1997, ModernApplied Statistics with s-plus, Springer, N.Y. Quadratic discriminantanalysis (QDA) takes the same input parameters and returns the sameresults as LDA. QDA uses quadratic equations, rather than linearequations, to produce results. LDA and QDA are interchangeable, andwhich to use is a matter of preference and/or availability of softwareto support the analysis. Logistic regression takes the same inputparameters and returns the same results as LDA and QDA.

Decision Trees

In some embodiments of the present invention, decision trees are used toclassify subjects using expression data for a selected set of molecularbiomarkers of the invention. Decision tree algorithms belong to theclass of supervised learning algorithms. The aim of a decision tree isto induce a classifier (a tree) from real-world example data. This treecan be used to classify unseen examples which have not been used toderive the decision tree.

A decision tree is derived from training data. An example containsvalues for the different attributes and what class the example belongs.In one embodiment, the training data is expression data for acombination of genes described in the present invention across thetraining population.

Clustering

In some embodiments, the expression values for a selected set ofmolecular markers of the invention are used to cluster a training set.For example, consider the case in which ten gene biomarkers described inone of the genes of the present invention are used. Each member m of thetraining population will have expression values for each of the tenbiomarkers. Such values from a member m in the training populationdefine the vector: Those members of the training population that exhibitsimilar expression patterns across the training group will tend tocluster together. A particular combination of genes of the presentinvention is considered to be a good classifier in this aspect of theinvention when the vectors cluster into the trait groups found in thetraining population. For instance, if the training population includespatients with good or poor prognosis, a clustering classifier willcluster the population into two groups, with each group uniquelyrepresenting either a deregulated IL-6/STAT3 signalling pathway statusor a regulated IL-6/STAT3 signalling pathway status.

Clustering is described on pages 211-256 of Duda and Hart, PatternClassification and Scene Analysis, 1973, John Wiley & Sons, Inc., NewYork. As described in Section 6.7 of Duda, the clustering problem isdescribed as one of finding natural groupings in a dataset. To identifynatural groupings, two issues are addressed. First, a way to measuresimilarity (or dissimilarity) between two samples is determined. Thismetric (similarity measure) is used to ensure that the samples in onecluster are more like one another than they are to samples in otherclusters. Second, a mechanism for partitioning the data into clustersusing the similarity measure is determined.

Similarity measures are discussed in Section 6.7 of Duda, where it isstated that one way to begin a clustering investigation is to define adistance function and to compute the matrix of distances between allpairs of samples in a dataset. If distance is a good measure ofsimilarity, then the distance between samples in the same cluster willbe significantly less than the distance between samples in differentclusters. However, as stated on page 215 of Duda, clustering does notrequire the use of a distance metric. For example, a nonmetricsimilarity function s(x, x′) can be used to compare two vectors x andx′. Conventionally, s(x, x′) is a symmetric function whose value islarge when x and x′ are somehow “similar”. An example of a nonmetricsimilarity function s(x, x′) is provided on page 216 of Duda.

Once a method for measuring “similarity” or “dissimilarity” betweenpoints in a dataset has been selected, clustering requires a criterionfunction that measures the clustering quality of any partition of thedata. Partitions of the data set that extremize the criterion functionare used to cluster the data. See page 217 of Duda. Criterion functionsare discussed in Section 6.8 of Duda. More recently, Duda et al.,Pattern Classification, 2nd edition, John Wiley & Sons, Inc. New York,has been published. Pages 537-563 describe clustering in detail. Moreinformation on clustering techniques can be found in Kaufman andRousseeuw, 1990, Finding Groups in Data: An Introduction to ClusterAnalysis, Wiley, New York, N.Y.; Everitt, 1993, Cluster analysis (3ded.), Wiley, New York, N.Y.; and Backer, 1995, Computer-AssistedReasoning in Cluster Analysis, Prentice Hall, Upper Saddle River, N.J.Particular exemplary clustering techniques that can be used in thepresent invention include, but are not limited to, hierarchicalclustering (agglomerative clustering using nearest-neighbor algorithm,farthest-neighbor algorithm, the average linkage algorithm, the centroidalgorithm, or the sum-of-squares algorithm), k-means clustering, fuzzyk-means clustering algorithm, and Jarvis-Patrick clustering.

Principal Component Analysis

Principal component analysis (PCA) has been proposed to analyze geneexpression data. Principal component analysis is a classical techniqueto reduce the dimensionality of a data set by transforming the data to anew set of variable (principal components) that summarize the featuresof the data. See, for example, Jolliffe, 1986, Principal ComponentAnalysis, Springer, N.Y. Principal components (PCs) are uncorrelate andare ordered such that the kth PC has the kth largest variance among PCs.The kth PC can be interpreted as the direction that maximizes thevariation of the projections of the data points such that it isorthogonal to the first k-1 PCs. The first few PCs capture most of thevariation in the data set. In contrast, the last few PCs are oftenassumed to capture only the residual ‘noise’ in the data.

PCA can also be used to create a classifier in accordance with thepresent invention. In such an approach, vectors for a selected set ofmolecular biomarkers of the invention can be constructed in the samemanner described for clustering above. In fact, the set of vectors,where each vector represents the expression values for the select genesfrom a particular member of the training population, can be considered amatrix. In some embodiments, this matrix is represented in a Free-Wilsonmethod of qualitative binary description of monomers (Kubinyi, 1990, 3DQSAR in drug design theory methods and applications, Pergamon Press,Oxford, pp 589-638), and distributed in a maximally compressed spaceusing PCA so that the first principal component (PC) captures thelargest amount of variance information possible, the second principalcomponent (PC) captures the second largest amount of all varianceinformation, and so forth until all variance information in the matrixhas been accounted for.

Then, each of the vectors (where each vector represents a member of thetraining population) is plotted. Many different types of plots arepossible. In some embodiments, a one-dimensional plot is made. In thisone-dimensional plot, the value for the first principal component fromeach of the members of the training population is plotted. In this formof plot, the expectation is that members of a first group will clusterin one range of first principal component values and members of a secondgroup will cluster in a second range of first principal componentvalues.

In one example, the training population comprises two classificationgroups. The first principal component is computed using the molecularbiomarker expression values for the select genes of the presentinvention across the entire training population data set where theclassification outcomes are known. Then, each member of the training setis plotted as a function of the value for the first principal component.In this example, those members of the training population in which thefirst principal component is positive represent one classificationoutcome and those members of the training population in which the firstprincipal component is negative represent the other classificationoutcome. In some embodiments, the members of the training population areplotted against more than one principal component. For example, in someembodiments, the members of the training population are plotted on atwo-dimensional plot in which the first dimension is the first principalcomponent and the second dimension is the second principal component. Insuch a two-dimensional plot, the expectation is that members of eachsubgroup represented in the training population will cluster intodiscrete groups. For example, a first cluster of members in thetwo-dimensional plot will represent subjects in the first classificationgroup, a second cluster of members in the two-dimensional plot willrepresent subjects in the second classification group, and so forth.

In some embodiments, the members of the training population are plottedagainst more than two principal components and a determination is madeas to whether the members of the training population are clustering intogroups that each uniquely represents a subgroup found in the trainingpopulation. In some embodiments, principal component analysis isperformed by using the R mva package (Anderson, 1973, Cluster Analysisfor applications, Academic Press, New York 1973; Gordon, Classification,Second Edition, Chapman and Hall, CRC, 1999.). Principal componentanalysis is further described in Duda, Pattern Classification, SecondEdition, 2001, John Wiley & Sons, Inc.

Nearest Neighbor Classifier Analysis

Nearest neighbor classifiers are memory-based and require no model to befit. Given a query point x0, the k training points x(r), r, . . . , kclosest in distance to x0 are identified and then the point x0 isclassified using the k nearest neighbors. Ties can be broken at random.In some embodiments, Euclidean distance in feature space is used todetermine distance as:

d(i)=·parallel·x(i)−xo·parallel.

Typically, when the nearest neighbor algorithm is used, the expressiondata used to compute the linear discriminant is standardized to havemean zero and variance 1. In the present invention, the members of thetraining population are randomly divided into a training set and a testset. For example, in one embodiment, two thirds of the members of thetraining population are placed in the training set and one third of themembers of the training population are placed in the test set. Profilesof a selected set of molecular biomarkers of the invention representsthe feature space into which members of the test set are plotted. Next,the ability of the training set to correctly characterize the members ofthe test set is computed. In some embodiments, nearest neighborcomputation is performed several times for a given combination of genesof the present invention. In each iteration of the computation, themembers of the training population are randomly assigned to the trainingset and the test set. Then, the quality of the combination of genes istaken as the average of each such iteration of the nearest neighborcomputation. The nearest neighbor rule can be refined to deal withissues of unequal class priors, differential misclassification costs,and feature selection. Many of these refinements involve some form ofweighted voting for the neighbors. For more information on nearestneighbor analysis, see Duda, Pattern Classification, Second Edition,2001, John Wiley & Sons, Inc; and Hastie, 2001, The Elements ofStatistical Learning, Springer, N.Y.

Evolutionary Methods

Inspired by the process of biological evolution, evolutionary methods ofclassifier design employ a stochastic search for an optimal classifier.In broad overview, such methods create several classifiers—apopulation—from measurements of gene products of the present invention.Each classifier varies somewhat from the other. Next, the classifiersare scored on expression data across the training population. In keepingwith the analogy with biological evolution, the resulting (scalar) scoreis sometimes called the fitness. The classifiers are ranked according totheir score and the best classifiers are retained (some portion of thetotal population of classifiers). Again, in keeping with biologicalterminology, this is called survival of the fittest. The classifiers arestochastically altered in the next generation—the children or offspring.Some offspring classifiers will have higher scores than their parent inthe previous generation, some will have lower scores. The overallprocess is then repeated for the subsequent generation: The classifiersare scored and the best ones are retained, randomly altered to give yetanother generation, and so on. In part, because of the ranking, eachgeneration has, on average, a slightly higher score than the previousone. The process is halted when the single best classifier in ageneration has a score that exceeds a desired criterion value. Moreinformation on evolutionary methods is found in, for example, Duda,Pattern Classification, Second Edition, 2001, John Wiley & Sons, Inc.

Bagging, Boosting and the Random Subspace Method

Bagging, boosting and the random subspace method are combiningtechniques that can be used to improve weak classifiers. Thesetechniques are designed for, and usually applied to, decision trees. Inaddition, Skurichina and Duin provide evidence to suggest that suchtechniques can also be useful in linear discriminant analysis.

In bagging, one samples the training set, generating random independentbootstrap replicates, constructs the classifier on each of these, andaggregates them by a simple majority vote in the final decision rule.See, for example, Breiman, 1996, Machine Learning 24, 123-140; and Efron& Tibshirani, An Introduction to Bootstrap, Chapman & Hall, New York,1993.

In boosting, classifiers are constructed on weighted versions of thetraining set, which are dependent on previous classification results.Initially, all objects have equal weights, and the first classifier isconstructed on this data set. Then, weights are changed according to theperformance of the classifier. Erroneously classified objects (molecularbiomarkers in the data set) get larger weights, and the next classifieris boosted on the reweighted training set. In this way, a sequence oftraining sets and classifiers is obtained, which is then combined bysimple majority voting or by weighted majority voting in the finaldecision. See, for example, Freund & Schapire, “Experiments with a newboosting algorithm,” Proceedings 13th International Conference onMachine Learning, 1996, 148-156.

In some embodiments, modifications of Freund and Schapire, 1997, Journalof Computer and System Sciences 55, pp. 119-139, are used. For example,in some embodiments, feature pre-selection is performed using atechnique such as the nonparametric scoring methods of Park et al.,2002, Pac. Symp. Biocomput. 6, 52-63. Feature pre-selection is a form ofdimensionality reduction in which the genes that discriminate betweenclassifications the best are selected for use in the classifier. Then,the LogitBoost procedure introduced by Friedman et al., 2000, Ann Stat28, 337-407 is used rather than the boosting procedure of Freund andSchapire. In some embodiments, the boosting and other classificationmethods of Ben-Dor et al., 2000, Journal of Computational Biology 7,559-583 are used in the present invention. In some embodiments, theboosting and other classification methods of Freund and Schapire, 1997,Journal of Computer and System Sciences 55, 119-139, are used.

In the random subspace method, classifiers are constructed in randomsubspaces of the data feature space. These classifiers are usuallycombined by simple majority voting in the final decision rule. See, forexample, Ho, “The Random subspace method for constructing decisionforests,” IEEE Trans Pattern Analysis and Machine Intelligence, 1998;20(8): 832-844.

Random Forest

Random Forest classifiers are an ensemble classifier that consists ofmany decision trees and outputs the class that is the mode of theclasses output by individual trees. Random Forests utilize bootstrappinginstead of cross-validation. For each iteration, a random sample (withreplacement) is drawn and the largest tree possible is grown. Each treereceives a vote in the final class prediction. To fit a random forest,the number of trees (e.g. bootstrap iterations) is specified. The randomforest algorithm gauges biomarker importance by the average reduction inthe training accuracy. The random forest method uses a number ofdifferent decision trees. A biomarker is considered to havediscriminating significance if it served as a decision branch of adecision tree from a significant random forest analysis.

Random forest (or random forests) is an ensemble classifier thatconsists of many decision trees and outputs the class that is the modeof the classes output by individual trees. (Breiman, Leo (2001). “RandomForests”. Machine Learning 45 (1): 5-32). Random forest is one of themost accurate learning algorithms available, i.e., produces a highlyaccurate classifier for data sets. (Caruana, Rich; Karampatziakis,Nikos; Yessenalina, Ainur (2008). “An empirical evaluation of supervisedlearning in high dimensions.” Proceedings of the 25th InternationalConference on Machine Learning (ICML)). The method combines “bagging”and the random selection of features in order to construct a collectionof decision trees with controlled variation. The selection of a randomsubset of features is an example of the random subspace method, which isa way to implement stochastic discrimination. Bootstrap distribution isused as a way to estimate the variation in a statistics based on theoriginal data. For each tree grown on a bootstrap sample, e.g., 150 or500, the error rate for observations left out of the bootstrap sample ismonitored. This is called the “out-of-bag” error rate.

Each tree is constructed using the following algorithm: (1) Let thenumber of training cases be N, and the number of variables in theclassifier be M; (2) The number m of input variables to be used todetermine the decision at a node of the tree; m should be much less thanM; (3) Choose a training set for this tree by choosing n times withreplacement from all N available training cases (i.e., take a bootstrapsample), and use the rest of the cases to estimate the error of thetree, by predicting their classes; (4) For each node of the tree,randomly choose m variables on which to base the decision at that node.Calculate the best split based on these m variables in the training set;and (5) Each tree is fully grown and not pruned (as may be done inconstructing a normal tree classifier).

For prediction a new sample is pushed down the tree. It is assigned thelabel of the training sample in the terminal node it ends up in. Thisprocedure is iterated over all trees in the ensemble, and the mode voteof all trees is reported as random forest prediction.

In one embodiment, random forest analysis involving classification andregression based on a forest of trees using random inputs is performedusing “randomForest: Breiman and Cutler's random forests forclassification and regression” (Depends: R (>=2.5.0), stats) (Version:4.6-6) (2012-01-06) (Fortran original by Leo Breiman and Adele Cutler, Rport by Andy Liaw and Matthew Wiener). See, A. Liaw and M. Wiener(2002). Classification and Regression by randomForest. R News 2(3),18-22.

Random Forests are further described in Liaw and Wiener, R News Vol.2/3, December 2002, pgs. 18-22; Dfaz-Uriarte and Alvarez, BMCBioinformatics. 2006 Jan. 6; 7:3); Statnikov et al., BMC Bioinformatics.2008 Jul. 22; 9:319; Shi et al., Mod Pathol. 2005 April; 18(4):547-57,Breiman, 1999, “Random Forests—Random Features,” Technical Report 567,Statistics Department, U.C. Berkeley, September 1999, which is herebyincorporated by reference in its entirety, each of which is incorporatedby reference herein it its entirety.

Other Algorithms

The pattern classification and statistical techniques described aboveare merely examples of the types of models that can be used to constructa model for classification. Moreover, combinations of the techniquesdescribed above can be used. Some combinations, such as the use of thecombination of decision trees and boosting, have been described.However, many other combinations are possible. In addition, in othertechniques in the art such as Projection Pursuit and Weighted Voting canbe used to construct a classifier.

As discussed in the Experimental Section, expression of the subjectbiomarker genes is preferably determined after real-time PCR using SYBRGreen, and a ΔΔCT method employed to analysis the data. The average CTvalues of house keeping genes in each sample is calculated as housekeeping gene CT value for that sample. ΔCT was calculated by subtractinghouse keeping CT value from individual assay CT value of same sample.ΔΔCT value was derived by further subtracting ΔCT value of controlsamples of each assay from its corresponding ΔCT value of treatmentsample. A Random Forest method is preferably used to analyze the ΔΔCTvalues of the samples and the expression thereof used to assess theregulatory status of the IL-6/STAT3 pathway activity in the sample.

Experimental Methods Used to Identify Inventive Il-6/Stat3 Signaling(16) Gene Signature

Identification of IL-6/STAT3 Response Genes by Gene Expression Profiling

The protocol that was used to identify the subject gene signature isdepicted schematically in FIG. 1. As depicted therein, human liverhepatocellular cells HepG2 and human normal mammary epithelial cellsMCF10A were plated in E-well plate in a density of 4×10⁵ cells per wellfor HepG2 and 2.5×10⁵ cells per well for MCF10A in 2 ml growth mediumand were reverse transfected with siRNA specifically targeting STAT3 ornon targeting siRNA as control at the time of plating cells. For eachwell, 6 ul of SureFECT transfection reagent (SABiosciences, a QIAGENcompany) was diluted into 400 μl of OptiMEM medium (Invitrogen). Thediluted transfection reagent was mixed with 40 nM STAT3 targeting siRNAduplex 7 (QIAGEN) or non-targeting AllStars siRNA (QIAGEN) as control.After incubation at room temperature for 20 min, the transfectionmixture was added into 6-well plate and covered with 2 ml growth mediumwith HepG2 or MCF10A cells. Plated cells were incubated in a cellculture incubator at 37 degrees C. with 5% CO2 supplied. Forty-eighthours after transfection, the medium with transfection mixture wasreplaced with 1 ml serum-free medium in each well and cells wereincubated for 16 hours in serum-free medium. After 16 hours serumstarvation, 1 ml serum-free medium with either 60 ng/ml recombinant IL-6(R&D Systems) or PBS as control was added on top of 1 ml cells bringingthe final serum-free medium volume to 2 ml and IL-6 concentration to 30ng/ml.

At the end of the 8 hour IL-6 treatment cells were lysed in 200 ul ofRLT Plus buffer (QIAGEN) for each well. The lysates were furtherprocessed to RNA isolation with RNeasy Plus RNA Isolation Kit fromQIAGEN according to manufacturer's protocol. (See Appendix attached tothis patent application) as described in experimental protocol section.At the end of isolation, 30 ul of RNase-free water was added to spincolumn to elute RNA off column. The concentration of RNA was measuredwith Nanodrop spectrophotometer (Thermo SCIENTIFIC) and the RNA wasfurther processed for real-time RT-PCR or microarray gene expressionprofiling analysis.

Real-time RT-PCR was employed to confirm the effect of IL-6 treatmentand STAT3 siRNA knockdown by measuring mRNA expression levels ofIL-6/STAT3 target genes and STAT3 itself respectively. 1 μg of total RNAwas reverse transcribed with RT2 First Strand cDNA synthesis kit(QIAGEN) according to protocol described in common experimental protocolsection. The 200 cDNA reaction was diluted to 100 μl of water forreal-time PCR analysis. For each real-time PCR reaction mixture, 1 μl ofcDNA was mixed with 10 of 10 μM primer mixture (forward and reverseprimers mixed), 12.5 μl of real-time PCR master mixture (QIAGEN) and10.5 ul water to a total volume of 25 μl. The primer sequences used forSOCS3, JUNB, BCL3, ZFP36, CEBPD, PIM1 are in the Table below:

Gene Symbol Refseq Forward Primer Reverse Primer SOCS3 NM_003955CCA CCT ACT GAA CCC TCC TCC TCT TCC GAC AGA GAT GCT GAA JUNB NM_002229CGA CTA CAA ACT CCT GAA ACC G GAA GAG GCG AGC TTG AGA GAC BCL3 NM_005178CAC TCT CTA CCA GAT AAC TGA GGA G TAA TAA TTT ACA TCG TGA TCC GTG CCEBPD NM_005195 CGC CAT GTA CGA CGA CGA G CGC CTT GTG ATT GCT GTT GZFP36 NM_003407 GCT ATG TCG GAC CTT CTC AG CTT CGC TAG GGT TGT GGA TGPIM1 NM_002648 GAT CCG CTA CCA TCG CTC C ATC TCC ACA CAC CAT ATC ATA CAG

The reaction mixture was added into 384-well real-time PCR plate induplicate wells with 10 μl each well. The PCR plate was sealed withoptical adhesive film (Applied Biosystems) and centrifuged for 2 minutesat 2000 rpm. The real-time PCR was run in ABI 7900 real-time PCR machine(Applied Biosystems) with PCR program as following, 95 degrees C. for 10min, 40 cycles of 95 degrees C. 15 seconds and 60 degrees C. 1 minutesfollowing melting curve analysis. The effect of IL-6 treatment wasconfirmed by upregulation of IL-6/STAT3 target genes such as SOCS3, JUNBand CEBPD in IL-6 treated samples compared to untreated samples (SeeFIG. 2B). The siRNA knockdown of STAT3 was verified by decreased mRNAexpression levels of STAT3 (See FIG. 2A).

After the confirmation of IL-6 treatment and STAT3 siRNA knockdown, RNAsamples were processed to whole genome microarray gene profilinganalysis. The 12 samples were split into four treatment groups intriplicates, sinon-no IL-6, sinon-IL-6, siSTAT3-no IL-6 andsiSTAT3-IL-6. 300 ng of total RNA was amplified and labeled withTargetAmp Nano-g Biotin-aRNA Labeling Kit (Epicentre Biotechnologies)according to manufacturer's protocol.

The amplification and labeling reagents and reaction parameters forHepG2 and MCF10A cell samples are shown below.

HepG2 Cells

RNA RNA T7- 1-strand cDNA con amount Oligo(dT) synthesis sample ng/ul300 ng H2O primer total HepG2/nc/+IL-6/1 1287.45 0.23 1.77 1 3.00HepG2/nc/+IL-6/2 1028.62 0.29 1.71 1 3.00 HepG2/nc/+IL-6/3 771.28 0.391.61 1 3.00 HepG2/nc/−IL-6/1 964.86 0.31 1.69 1 3.00 HepG2/nc/−IL-6/2547.48 0.55 1.45 1 3.00 HepG2/nc/−IL-6/3 692.68 0.43 1.57 1 3.00HepG2/Stat3/+IL-6/1 975.75 0.31 1.69 1 3.00 HepG2/Stat3/+IL-6/2 1026.540.29 1.71 1 3.00 HepG2/Stat3/+IL-6/3 1072.54 0.28 1.72 1 3.00HepG2/Stat3/−IL-6/1 814.68 0.37 1.63 1 3.00 HepG2/Stat3/−IL-6/2 616.460.49 1.51 1 3.00 HepG2/Stat3/−IL-6/3 636.18 0.47 1.53 1 3.00incubate 65 degrees C. for 5 min, chill on ice 1 min, centrifuge briefly

1st strand cDNA synthesis master mix 14 1st strand cDNA premix 21 DTT3.5 superscript III (200 u/ul) 3.5 Total 28 gently mixadd 2 ul to each reaction, gently mix, incubate 50 degrees C. for 30min.Second Strand cDNA Synthesis

second strand cDNA synthesis master mix 13 2nd-strand cDNA premix 58.52nd-strand DNA polymerase 6.5 total 65 gently mixadd 5 ul to each reaction, gently mix, incubate 65 degrees C. 10 min,centrifuge briefly.incubate at 80 degrees C. for 3 min, centrifuge briefly, chill on ice.

In Vitro Transcription of Biotin-aRNA

warm T7 RNA polymerase to RT and thaw other reagent at RT

in vitro transcription master 13 mix setup at RT T7 transcription buffer26 UTP/biotin-UTP 39 NTP premix 130 DTT 39 T7 RNA polymerase 26 Total260 gently mixadd 20 ul to each reaction, gently mix, incubate 42 degrees C. for 4 h(don't exceed 4 h)add 2 ul Rnase-free Dnase I to each reaction, mix gently, incubate 37degrees C. 15 min.Biotin-aRNA Purification (SABio cRNA Cleanup Kit)Bind aRNA to Spin Columna. transfer entire reaction (32 ul) to 1.5 ml tubeb. add 112 ul lysis & binding buffer (G6) to each reaction, mix bypipetting 2-3×c. add 112 ul RT 100% ETOH, mix by pipettting 5-6×d. immediately load on spin columne. centrifuge 8000 g for 30 secf. discard flow-through, put column back to collection

Washing Spin Column

a. add 400-500 ul washing buffer (017-FETOH) to each spin columnb. centrifuge 8000 g 30 secc. discard flow-through, put column back to collection tubed. add 200 ul washing buffer (017+ETOH) to each spin columne. centrifuge 11000 g 1 minf. discard flow-through, put column back to collection tubeg. centrifuge 11000 g 2 min (180 degree rotate from previousorientation)Elute aRNA from Spin

Column

a. transfer spin column to new elution tubeb. add 40 ul (<40 ug, 80 ul if >40 ug) H₂O into column

c. sit in RT 2 min

d. centrifuge 8000 g for 1 mine. store aRNA −80 degrees C.

MCF10A Cells

RNA RNA T7- 1-strand cDNA con amount Oligo(dT) synthesis sample ng/ul300 ng H2O primer Total MCF10A/nc/+IL-6/1 839.39 0.36 1.64 1 3.00MCF10A/nc/+IL-6/2 984.02 0.30 1.70 1 3.00 MCF10A/nc/+IL-6/3 875.17 0.341.66 1 3.00 MCF10A/nc/−IL-6/1 798.19 0.38 1.62 1 3.00 MCF10A/nc/−IL-6/2626.77 0.48 1.52 1 3.00 MCF10A/nc/−IL-6/3 445.07 0.67 1.33 1 3.00MCF10A/Stat3/+IL-6/1 760.52 0.39 1.61 1 3.00 MCF10A/Stat3/+IL-6/2 735.990.41 1.59 1 3.00 MCF10A/Stat3/+IL-6/3 748.25 0.40 1.60 1 3.00MCF10A/Stat3/−IL-6/1 629.8 0.48 1.52 1 3.00 MCF10A/Stat3/−IL-6/2 406.170.74 1.26 1 3.00 MCF10A/Stat3/−IL-6/3 517.99 0.58 1.42 1 3.00incubate 65 degree C. for 5 min, chill on ice 1 min, centrifuge briefly

1st strand cDNA synthesis master mix 14 1st strand cDNA premix 21 DTT3.5 superscript III (200 u/ul) 3.5 Total 28 gently mixadd 2 ul to each reaction, gently mix, incubate 50 degrees C. for 30min.Second Strand cDNA Synthesis

second strand cDNA synthesis master mix 13 2nd-strand cDNA premix 58.52nd-strand DNA polymerase 6.5 total 65 gently mixadd 5 ul to each reaction, gently mix, incubate 65 degrees C. 10 min,centrifuge briefly.incubate at 80 degrees C. for 3 min, centrifuge briefly, chill on ice.

In Vitro Transcription of Biotin-sRNA

warm T7 RNA polymerase to RT and thaw other reagent at RT

in vitro transcription master 13 mix setup at RT T7 transcription buffer26 UTP/biotin-UTP 39 NTP premix 130 DTT 39 T7 RNA polymerase 26 Total260 gently mixadd 20 ul to each reaction, gently mix, incubate 42 degrees C. for 4 h(don't exceed 4 h)add 2 ul Rnase-free Dnase I to each reaction, mix gently, incubate 37degrees C. 15 min.Biotin-aRNA Purification (SABio cRNA Cleanup Kit)Bind aRNA to Spin Columna. transfer entire reaction (32 ul) to 1.5 ml tubeb. add 112 ul lysis & binding buffer (G6) to each reaction, mix bypipetting 2-3xc. add 112 ul RT 100% ETOH, mix by pipettting 5-6×d. immediately load on spin columne. centrifuge 8000 g for 30 secf. discard flow-through, put column back to collection

Washing Spin Column

a. add 400-500 ul washing buffer (G17+ETOH) to each spin columnb. centrifuge 8000 g 30 secc. discard flow-through, put column back to collection tubed. add 200 ul washing buffer (G17+ETOH) to each spin columne. centrifuge 11000 g 1 minf. discard flow-through, put column back to collection tubeg. centrifuge 11000 g 2 min (180 degree rotate from previousorientation)Elute aRNA from Spin

Column

a. transfer spin column to new elution tubeb. add 40 ul (<40 ug, 80 ul if >40 ug) H₂O into column

c. sit in RT 2 min

d. centrifuge 8000 g for 1 mine. store aRNA −80 degrees C.

The concentration of labeled antisense RNA was measured with Nanodropspectrophotometer (Thermo SCIENTIFIC). Total 750 ng of labeled antisenseRNA was hybridized onto an Illumina Human HT-12 BeadChip (Illumina)according to the manufacturer's standard protocol for 12 samples chip(Illumina Whole Genome Gene Expression Direct Hybridization Assay).

Hybridized BeadChip was washed and scanned on an iScan (Illumina)according to manufacturer's standard protocol. The image file wasprocessed with GenomeStudio software (Illumina) without backgroundcorrection and normalization. The sample probe expression file wasexported as GeneSpring format for further analysis with GeneSpringsoftware (Agilent). The expression data was analyzed with GeneSpringwith its guided workflow and fold changes and statistical analysis wascomputed between groups during the guided workflow analysis.

After effecting these protocols, three gene lists were selected from theidentified IL-6 response genes as IL-6/STAT3 response genes. Two genelists (HepG2 list 1 and HepG2 list 2) were derived from HepG2 cell withdifferent selection criteria. HepG2 list 1 had 57 genes (66 probes) andwas selected based on sinon-IL-6 vs sinon-no IL-6 adjusted P<=0.05,fold>=1.5 and sinon-IL-6 vs siSTAT3-IL-6 P<=0.05 (See FIG. 3A). HepG2list 2 had 52 genes (55 probes) and was selected based on sinon-IL-6 vssinon adjusted P<=0.05, and sinon-IL-6 vs siSTAT3-IL-6 adjusted P<=0.05(see FIG. 3B). 14 genes were derived from MCF10A samples and wereselected based on sinon-IL-6 vs sinon-no IL-6 P<=0.05, fold>=1.5, andsinon-IL-6 vs siSTAT3-IL-6 P<=0.05. All three gene lists showed similarpattern of expression changes across treatment conditions. Expressionchanges of those genes were statistically significant in response toIL-6 and were reversed upon treatment with STAT3 siRNA. A list of 84genes from all three gene lists combined were selected as IL-6/STAT3response genes from microarray studies. These 84 genes plus 4 well knownIL-6/STAT3 target genes and 8 house keeping genes were converted toreal-time PCR platform for further verification.

Identification of IL-6/STAT3 Gene Expression Signature

To test these 88 IL-6/STAT3 response genes with real-time PCR, SYBRgreen based real-time PCR assay was designed for each individual gene.The sequence information for all primers is contained in FIG. 6.

Sixteen samples were employed to test the expression of these 88 genes.The IL-6/STAT3 pathway activity was negatively regulated in sevensamples with STAT3 siRNA treatment. In contrast, nine samples had theirIL-6/STAT3 pathway activity positively regulated and they werestimulated with IL-6 to activate IL-6/STAT3 pathway activity.

The STAT3 siRNA was reverse transfected into HepG2, 293H, Hela, A549,U105MG, HT1080 and MDA-MB-231 cells. For each well of 6-well plate, 6 μlof Surel-ECT transfection reagent (SABiosciences, a QIAGEN Company) wasdiluted into 200 μl of OptiMEM medium (Invitrogen). The dilutedtransfection reagent was mixed with 40 nM STAT3 targeting siRNA duplex7, or non-targeting siRNA (QIAGEN) as control. Master transfectionmixture for 4 wells was prepared for either STAT3 or non-target siRNA.After incubation at room temperature for 20 minutes, 200 μl oftransfection mixture was added into each well in eight 6-well plateswith one plate for each cell line including HepG2, 293H, Hela, A549,U105MG, HT1080 and MDA-MB-231. Each plate had two wells containing STAT3siRNA mixture and two wells containing non-target siRNA mixture. Thesetwo duplicate wells were for protein extraction and RNA isolationrespectively. During the 20 minute incubation time, different cell lineswere trypsinized, washed off plate and resuspended in 8 ml culturemedium and cell numbers were counted with a hemocytometer.

Cells were diluted into culture medium in a concentration of 1-2×10⁵cells per ml. For each well, 2 ml of cells (2-4×10⁵) were plated in6-well plate on top of 200 μl transfection mixture and the plate wasmixed well. The cell culture plates were put back into incubator andincubated for 72 hours at 37 degrees C. with 5% CO2 supplied. At the endof 72 hours incubation, cells were either lysed in 50 μl modified RIPAbuffer for protein lysate extraction or in 200 μl lysis RLT Plus bufferfor RNA isolation. The protein extraction and western blot was carriedout according to western blot protocol in common experimental protocolsection with rabbit anti-pSTAT3 (1:1000) and rabbit anti-STAT3 (1:1000)antibody. The decreased STAT3 protein levels in both phosphorylated andtotal forms verified the effect of STAT3 siRNA (See FIG. 4B). The RNAwas isolated with RNeasey Plus RNA Isolation Kit from QIAGEN accordingto manufacturer's protocol as described in experimental protocolsection.

To obtain nine positively regulated samples with IL-6 treatment, ninedifferent cell lines were plated in 6-well plates in a density of2-4×10⁵ cells/well/2 ml. After 24 h of plating, cells were switched toserum-free medium by removing normal culture medium, washing cells inPBS two times and replacing with 1 ml serum-free medium each well. After16 hours in serum-free medium, cells were replaced with serum-freemedium with or without 30 ng/ml IL-6 in duplicate wells for anadditional incubation of 8 h. At the end of 8 hours incubation, cellswere either lysed in 50 μl modified RIPA buffer for protein lysateextraction or in 200 μl RLT Plus lysis buffer for RNA isolation. Theprotein extraction and western blot was carried out according to westernblot protocol with rabbit anti-pSTAT3 (1:1000) and rabbit anti-STAT3(1:1000) antibody. The increased pSTAT3 protein levels confirmed theeffect of IL-6 (See FIG. 4A). The RNA was isolated with RNease Phis RNAIsolation Kit from QIAGEN according to manufacturer's protocol describedin experimental protocol section.

To verify 84 IL-6/STAT3 response genes with SYBR green based real-timePCR on the described seven negative and nine positive samples, 1 μg oftotal RNA was reversed transcribed with RT2 First Strand cDNA synthesiskit (SABiosciences, a QIAGEN company) according to manufacturer'sprotocol in experimental protocol section. The 20 μl of reversetranscription reaction was diluted to 200 μl with water. For eachreal-time PCR reaction, 1 μl of diluted cDNA was mixed with 5 μl of SYBRgreen PCR master mixture and 4 μl of water to give a final volume of 10μl of each reaction. A master mixture of 110 real-time PCR reactions wasprepared for each sample and added into 384-well plate with 10 μl foreach well. Each sample had 96 reactions in 96 wells corresponding to 96different PCR assays (88 IL-6/STAT3 response genes plus 8 house keepinggenes) and each 384-well plate was loaded with reactions for 4 samples(96×4 wells). The 384-well plates were run in ABI 7900 real-time PCRmachine (Applied Biosystems) with SYBR green based real-time PCR programas following, 95 degrees C. for 10 min, 40 cycles of 95 degrees C. 15seconds and 60 degrees C. 1 minutes following by melting curve analysis.

After real-time PCR, a ΔΔCT statistical analysis method was employed toanalysis the data. The average CT values of 8 house keeping genes ineach sample were calculated as house keeping gene CT value for thatsample. OCT was calculated by subtracting house keeping CT value fromindividual assay CT value of same sample. The ΔΔCT value was derived byfurther subtracting ΔCT value of control samples of each assay from itscorresponding ΔCT value of treatment sample.

A Random Forest classifier method was used to analyze the ΔΔCT values ofseven negative and nine positive samples. Up to 150 bootstrap samplescontaining 14 out of the 16 training samples were selected and thebootstrap was performed without replacement and stratified by class(selected 7 stimulated and 7 repressed). For each bootstrap, a randomforest classifier (using default parameters) was trained on the 14samples with all 88 gene expression measurements. Based on the randomforest variable importance measure (mean decrease in out-of-bagclassifier accuracy when a gene's expression values are randomlypermuted), the top 16 ranked genes were selected from each bootstrapprocess. Each 150 bootstrap iteration generated a slightly differentlyranked gene list and the average rank across the 150 bootstrapiterations for each gene was calculated. Genes were ranked by thisaverage rank, and the top 16 genes were select as the final signaturegene set. These sixteen genes were defined as a gene expressionsignature that differentially classified positive samples from negativesamples (see FIG. 5A). These 16 genes and their Accession numbers arelisted in the table below.

Gene Symbol RefSeq_ID STAT3 NM_213662 SOCS3 NM_003955 IFITM2 NM_006435CEBPD NM_005195 JUNB NM_002229 TUBB2A NM_001069 IL-6ST NM_002184 CASP4NM_001225 PROS1 NM_000313 TNFRSF1A NM_001065 PVRL2 NM_002856 PHF21ANM_016621 BCL3 NM_005178 NRP1 NM_003873 GLRX NM_002064 TGM2 NM_00461

The utility of the obtained 16 gene signature was verified on these 16samples by cross validation with Random Forest classification methodusing described bootstrap process. During each of 150 bootstrap process,the top 16 genes were used to train a new random forest classifier andthe model was used to score the two out-of-training samples. Theperformance of the classification method was estimated based on theability of the model to classify two out-of-training samples during eachbootstrap process. Using the described methods 14 out of 16 samples wereclearly classified correctly based on this 16 gene signature (See FIG.5B).

Standard Protocols for Common Experiments:

Cell Culture and Chemicals

All cell culture medium was purchased from Invitrogen and different celllines were purchased from ATCC. 293H, HepG2, U373MG, U105MG, andMDA-MB-231 cells were cultured in DMEM medium with 10% FBS, 1 mM sodiumpyruvate and non-essential amino acid (Invitrogen). CCD1079SK, BJ,IMR90, Hela, HT1082 and MCF7 cells were cultured in MEM medium with 10%FBS. Lncap and Raji cells were cultured in RPMI 1640 medium with 10%FBS. HT29 cells were cultured in McCoy's 5A modified medium with 10%FBS. All cells were cultured in a cell culture incubator at 37 degreesC. supplied with 5% CO2. All chemicals used in experiments were fromSigma unless indicated with other source.

Protocol for Cell Lysis and Western Blot

At the end of experimental treatment, cells were lysed in Modified RIPAbuffer (150 mM NaCl, 50 mM TrisHCl, 1% IGEPAL, 0.5% sodium deoxycholate,1 mM EDTA, 1% Triton X-100 and 0.1% SDS with protease and phosphataseinhibitor) (all chemicals from Sigma). For each well in 6-well plate,cell culture medium was aspirated and washed with 1 ml PBS. 50 μl ofModified RIPA buffer was added to each well and cells were scrapped offwells in Modified RIPA buffer. Cell lysate was transferred to 1.5 mlmicrocentrifuge tube and incubated on ice for 30 min. After 15 minutescentrifuge at 15000 rpm at 4 degrees C., supernatant was transferred toa new 1.5 ml tube and protein concentration was measured with BCAprotein assay according to manufacturer's standard protocol (Pierce).The cell lysate was diluted in 30 μl of H₂O to 2 μg/μl proteinconcentration and mixed with 30 μl of 2×SDS sample buffer (BioRAD) togive a final concentration of 1 μg/μl. The diluted lysate was heated at70 degrees C. for 10 minutes to denature the protein. The lysate wascentrifuged at 15000 rpm for 1 minutes after heating and was loaded on aprecast 4-12% NuPAGE Novex Bis-Tris Mini gel (Invitrogen) with 15 μllysate for each well. The gel was run at a constant voltage of 150 V for1.5 hours following transfer to a nitrocellulose membrane at a constantvoltage of 30 V for 2 hours according to manufacturer's protocol(Invitrogen). The nitrocellulose membrane was blocked in 5% milk inwestern blot wash buffer (1×PBS plus 0.1% Tween-20) for 1 hours at roomtemperature. Separate membranes were further incubated with rabbitanti-pSTAT3 (1:1000) (Cell Signaling), rabbit anti-STAT3 (1:1000) (CellSignaling) and rabbit anti-GAPDH (1:2000) (Cell Signaling) primaryantibodies at 40 C overnight. The next day, membranes were took out from40 C and further incubated at room temperature for 30 minutes followingthree times of wash in western blot wash buffer with 5 minutes for eachwash. Membranes were incubated with goat anti-rabbit (1:4000) secondaryantibody (Cell Signaling) for 1 hours at room temperature. Membraneswere washed in western blot wash buffer 5 minutes for three times. Todetect protein band on membranes, mixed western blot substrate (0.75 mlperoxide solution mixed with 0.75 ml luminol enhancer solution) (ThermoSCIENTIFIC) was added to each membrane and incubated at room temperaturefor 1 minutes to cover the entire membrane. The membrane was exposed toFuji image machine LAS-3000 (Fuji Film) for 2 minutes withchemiluminecence filter. The effect of IL-6 treatment was demonstratedby increased protein levels of pSTA3 in IL-6 treated samples compared tono treated samples. The effect of STAT3 siRNA was demonstrated bydecreased protein levels of phorylated and total STAT3 in STAT3 siRNAtransfected samples compared to non target siRNA transfected samples.

Total RNA Isolation with QIAGEN RNeasy Plus Mini Kit

To harvest cells grown in 6-well plate for RNA isolation, cell culturemedium was removed and 200 μl of RNeasy Plus buffer was added into eachwell. Cells were scrapped off plate and lysate was transferred to a 1.5ml microcentrifuge tube for immediate RNA isolation or stored at −80degrees C. to isolate RNA later. To isolate RNA, transfer thehomogenized lysate to a gDNA Eliminator spin column placed in a 2 mlcollection tube. Centrifuge for 30 s at ≧8000×g (≧10,000 rpm). Discardthe column, and save the flowthrough. One volume (200 μl) of 70% ethanolwas added to the flowthrough and mixed 6 times by pipetting. The mixedsample was added to an RNeasy spin column placed in a 2 ml collectiontube and centrifuged for 1 minutes at ≧8000×g (≧10,000 rpm). The columnwas washed with 700 μl of buffer RW1 by centrifuging for 1 minutes at≧8000×g (?_(—)10,000 rpm). Buffer RPE (500 μl) was added to the RNeasyspin column and centrifuged for 1 minutes at ≧8000×g (≧10,000 rpm) towash the spin column membrane. Another 500 μl Buffer RPE was added tothe RNeasy spin column and centrifuged for 2 minutes at ≧8000×g (≧10,000rpm) to wash the spin column membrane. The RNeasy spin column was placedin a new 2 ml collection tube and centrifuged at full speed for 1 min.RNeasy spin column was transferred to a new 1.5 ml collection tube and30 μl RNase-free water was directly added to the spin column membrane.The spin column was sit at room temperature for 2 minutes andcentrifuged for 1 minutes at ≧8000×g (≧10,000 rpm) to elute the RNA.

Protocol for Reverse Transcription with RT² EZ First Strand Kit (QIAGEN)

Total RNA of 300-1000 ng was diluted with RNase-free H₂O to 8 μl andmixed with 6 μl of GE2 (genomic DNA elimination) buffer. The reactionwas incubated at 37° C. for 5 min, and immediately placed on ice for 1minute. 6 μl of the BC5 (RT Master Mix) was added to each 14-μl GenomicDNA Elimination Mixture for a final volume of 20 μl. The reaction wasincubated at 42° C. for exactly 15 minutes and then immediately stoppedby heating at 95° C. for 5 minutes. Incubation at 37° C., 42° C. and 95°C. was done on a thermal cycle GenAmp PCR System 2700 (Applied Systems).The finished reaction was put on ice until ready to use for real-timePCR, or placed at −20° C. for long-term storage.

Protocol for Reverse Transcription with RT² First Strand Kit (QIAGEN)

Total RNA of 300-1000 ng was diluted with RNase-free H₂O to 8 μl andmixed with 2 μl of GE (genomic DNA elimination) buffer. The reaction wasincubated at 42° C. for 5 min, and immediately placed on ice for 1minute. 10 μl of the RT cocktail (4 μl BC3, 1 μl P2, 2 μl of RE3 and 3μl of H₂O) was added to each 10-μl Genomic DNA Elimination Mixture for afinal volume of 204 The reaction was incubated at 42° C. for exactly 15minutes and then immediately stop the reaction by heating at 95° C. for5 minutes. Incubation at 42° C. and 95° C. was done on a thermal cycleGenAmp PCR System 2700 (Applied Systems). The finished reaction was puton ice until ready to use for real-time PCR, or placed at −20° C. forlong-term storage.

Primers

The primers used to amplify the 88 response genes are listed in thetable below and provided in FIG. 6.

Gene symbol Primer_F Primer_R ACSL3 GGA GTG TTA GGA GCA GCC AGCAT ACG ATG TTT GTG ATG CAA C ADFP TCC TGT CCA ACA TCC AAG GTGTTG CTA GAA GTG AGG AGG CTG ARFGAP3 GTG AAA GGT GTT GCT GTT TGAAT GAC TGT TCT CCC ATA CAC G BTBD11 GTA TCC TCA GAG ATG CTG CGTTGA CAG AGA AAG CAC ACC AAA TG G C20orf46 CCT CCT TCC CAA TGG CAT CAGC TGC CCA GTC TCG TGT TC C8A TCA ATC CAT GAC CAG GGA GAAT GTT TCA GGT GTC TGC TTG CASP4 GAG AGA CAG CAC AAT GGG CTCCTT CCG AAA TAC TTC CTC TAG GTG CD14 CCA GAA CCT TGT GAG CTG GACCGC TTT AGA AAC GGC TCT AGG CEBPD CGC CAT GTA CGA CGA CGA GCGC CTT GTG ATT GCT GTT G CFB GCT CAC GCC CGA GAC TTT CAAC CCA AAT CCT CAT CTT GGA G CHI3L1 ACT CGG GAT TAG TAC ACA CTTGTT TGG CTC CTT GGT GAT AG GTT G CITED2 AAT GGG CGA GCA CAT ACA CGTG CCC TCC GTT CAC AGT C CXADR CAT AGG TGA AGA CAT GGG TGAGAG ACT GGT GGG CCA TAA ATA AC ATA G DNAJC12 GAA GGC AAA GGA GAT TCT GACACT GCT GGA ATG GCA TCG AC EFNA1 AGC TGA ATG ACT ACG TGG ACAACT GCC AGC GGA CTT GGT C TC FBN2 CAG ATC AGC CTA GAG AGT GTCCCT TTG GTG GAT GCG GAA G G FGA GAG ACT CCA CAT TTG AAA GCACTC TGA CAG GGC GAG ATT TAG AG FGB CAT GCA GCC AAT CCA AAC GTTC ATC CAT ACT ACA CCA TCA TC FILIP1L CTT CAA ATG CAG CCA GTC TACGAT CTC CAG GTT GCA CAA AG FKBP14 GAG GTT GCG GTA AGC CGA GGCC ACC AAT CAC TAG GAG C FLOT2 GGT GAA GCA GGT CCT CTT GCTT CAT CCG CTC AGC CTC TG FLRT3 CAG CCT GGA GCA TCT TCC TCCAG TAA ATG AAA CCC GCA TCG FVT1 ATG AGC ATC AAT TAC CTG GGCGGC TGT GAA ACC GAA TAA TC AG GALNAC4S- GAG GCT TTG ATG ACC AAG AGCGGC CTG TAG AAA TCC CGC AG 6ST GBP2 GAA CGT ATA AAG GCT GAA TCTTGA AGT TTA AGA GCG AGG GTC GC GK TGC ATG ACC CTC CAA GTA GACTAG AGG GAA TGG AGC AGG ATG GLRX GCA GAG GCT GTG GTC ATG CTGC TTT AAT CTT TGC TGG TAG TC GSDMC AGA GAT AGG GCT GTG CCT CTTA TAC ATA GTG AAA CGC TTA CGT C GSTT1 GCC AAG GAC TTC CCA CCT GAAT GCT TTG TGG ACT GCT GAG HAMP CCA TGT TCC AGA GGC GAA GGCA GCA CAT CCC ACA CTT TG HK1 AAC GTG TCC TTC CTC CTG TCCTG CTT GCC TCT GTG CGT AAC HP TAA GGC ATT ATG AAG GCA GCCCA GTC GCA TAC CAG GTG TC HP4 TAG GGC GTG TGG GTT ACG TGGTT CGT TCA GTA TGG GCT G IFITM2 TCC CAC GTA CTC TAT CTT CCACTG ATG CAG GAC TCG GCT G TTC IFNGR1 AGA ATG GAT TGA TGC CTG CTTG TCC AAC CCT GGC TTT AAC IFNGR2 GGA GCC TGT TTC TTC CTG GTCCTC TTC TAT CTG TAA TGG GAT GC IL1RAP CTA GAC ACC ATG AGG CAA ATCCCT AGT CCA ATA CCA GAT CAG AG INSIG2 TGG CAG AAG GAG AGA CAG AGCTC GAA TCA TCA AGT TCA CAC TC KLF9 GGC CGC CTA CAT GGA CTT CAGC TCT TGG CGA TGG TGA C LBP TGA GAG TTT GAG GAC AAG AAACGG AGC TGA GAG CAG AAA TG GAT G LOXL4 GTG ATG AAC GCC CAG CTA GTGCAG TCC GGC CCA GAT TGT AG LRG1 CTA GAA CTC TGT TCC TGC TGCCAG GTG GTT GAC AGG AGA TG LY96 GTT GTT GAA GCT ATT TCT GGGTTG AAT TAG GTT GGT GTA GGA TG AG MATN3 TCT CCC GGA TAA TCG ACA CTCCCT GAC ATG GTG CCT GTT GAC MBL2 TGA GGT TTC TAC TGG GAC CACCAG TTC TGC ATA AGT TGA TTG ATA G MOCOS GGC TGC TAT ATG ACC GGA GTCG ATG AAG GGC TGG ATC AG NEK6 GAA CCA CCC AAA TAT CAT CAACTG CGT CAG CCA ACT CCA G G NRP1 CAA CAA CTA TGA TAC ACC TGATTC CAC TTC ACA GCC CAG C GC ORM1 GCA TTT CGC TCA CTT GCT GAGT TCT TCT CAT CGT TCA CGT C P2RY5 TTG TAT GGG TGC ATG TTC AGCTGT AAG TTG TAG TTT CAT TTC GGA C PC CCT TTC AGC CAT CGT CCT TTCCCA CCT GAC CCA CCA CTT GTA G PFKFB3 GAG CCG CAT CGT GTA CTA CCTG GAG GTT GTG CTC GTT CTC PGK1 TCT GTT TGA TGA AGA GGG AGCCAG CCA GCA GGT ATG CCA G PHF21A GGC AGA AGG AGA TGC ACA GCTCA GAG TCT ACA GGT TTG GAG AG PLA2G2A TGT GTG AGT GTG ATA AGG CTGGAG AGG GAA ATT CAG CAC TGG C PLOD2 TAG CCG TAT ATC TGG TGG TTAGTG TAA CTG GTG CAA TGA ACT C TG PLSCR4 AAC TTG CTT CTG TTG CAC TTTTAC ATC CCA TTC TAC ATA CTG AG ACT G PROS1 ATC GGA TAC AGG CCC TAA GTCTTG TCC AAG ACG GCA AGT TG PVRL2 AAG CCA AAG AGA CTC AGG TGCAG GTA TCA GGG CTG GTT CCT C RAB43 GGC CAG GTG ATC TTC CTT AGCCTA GAC CAC AAA CCG ACG CAG RCN1 CAT GAG GAG AAT GGC CCT GCAT CTT TGT CTA ACT TCC CGT C RHOB TGC CAT AAG CGA ACT TTG TGCGTG TGG TCA GAA TGC TAC TGT C RNASE4 TGC AGA GGA CCC ATT CAT TGCAA GTT GCA GTA GCG ATC AC SEMA4B GGA GAA GCC ATG TGA GCA AGCCG TTG CGT AGC CAG AGT C SERPINA3 CTC TCA GTA AGG AAC TTG GAAAGA GCT ACA CAG GGA ATC GCT G TG SERPINB13 GAA AGA AAG GTG AAT CTG CACCAG GAA CTT CTG GGC GTA CAA C SERPINB3 CGC GGT CTC GTG CTA TCT GGGA AAG GGT GAT TAC AAT GGA AC SERPINE1 AGA GAC AGG CAG CTC GGA TTCCCA AAG TGC ATT ACA TCC ATC SLC17A2 TTG AGT CTG GTT GGA GGA ATGTCA GAG GCG GGT AAG GGT C SNX25 TCA GTG AGC AAA TGT TGG TTTCTT CTG ATT GTG GTC GGT G AC SOD2 GGA GCA CGC TTA CTA CCT TCCAT TCT CCC AGT TGA TTA CAT TC SPINK1 CTG AAG AGA CGT GGT AAG TGCCAC TGA GAA GAA AGA TGC CTG SPP1 CTG AAA CCC ACA GCC ACA AGTGA CTA TCA ATC ACA TCG GAA TG STAT3 TGA CAT GGA GTT GAC CTC GCTG GAA CCA CAA AGT TAG TAG TTT C TACC1 CCT GTG TCG GTG TCC TGT GAGG TGA GCA CGG CTG TCT TG TGM2 CTT CAC AAG GGC GAA CCA CGCG GCA GAC GTA CTC CTC AG TMEM166 CTG TAC TTT GTT TCT GGC GTGGTC GCT GCT GCT CTC TCT GTC TG TNFRSF1A TGT TAC ACT AAT AGA AAC TTGCCT TAG GAC AGT TCA GCT TGC GCA C TOX3 GAT TGT CAC ATC AGT CAC CATTTG CAC CGA AGG ACT CAC TTG TG TPST1 TGG ATG AGG CTG GTG TTA CTGATC TCG GAC CAT CAG GAG AAA TUBB2A AAC TTC TCA GAT CAA TCG TGCAGA CCA TGC TTG AGG ACA AC TUBB3 AAC AAC TGG GCC AAG GGT CGTC GGG ATA CTC CTC ACG CAC XBP1 ATA TCC TGT TGG GCA TTC TCGAA AGG GAG GCT GGT AAG GAA C ZNF684 AGG ACG GTA GCC GGT ATT CGCT CCA AGC CTG GGA TCA G IL6ST AAG ATT TGA AAC AGT TGG CATCCT TCA CTG AGG CAT GTA GC GGA G SOCS3 CCA CCT ACT GAA CCC TCC TCCTCT TCC GAC AGA GAT GCT GAA JUNB CGA CTA CAA ACT CCT GAA ACCGAA GAG GCG AGC TTG AGA GAC G BCL3 CAC TCT CTA CCA GAT AAC TGATAA TAA TTT ACA TCG TGA TCC GGA G GTG C B2M GCA AGG ACT GGT CTT TCT ATCACT TAA CTA TCT TGG GCT GTG AC TC HPRT1 GGC CAT CTG CTT AGT AGA GCTTA GGA ATG CAG CAA CTG AG RPL13A TGA GTG AAA GGG AGC CAG AAGTGC AGA GTA TAT GAC CAG GTG GAPDH AGA GCA CAA GAG GAA GAG AGAGGT TGA GCA CAG GGT ACT TTA G TTG ACTB AAT GCT TCT AGG CGG ACT ATGCTC CAA CCG ACT GCT GTC AC TFRC AGC TGA GAT TCC TGG TTC GCAT GCC CTG TAT TCA TAT TGT G HSP90AB1 GCA GAG GAA CCC AAT GCT GGGA CAC TAT ACA AGG GCA CAA G PPIA AAT GGG TTA CTT CTG AAA CATGAC TCC TAC CCT CAG GTG GTC CAC

1. A composition for the detection of the regulation status ofIL-6/STAT3 signaling pathway in a cell sample or subject, comprisingprimers that amplify at least 5 of the genes selected from the groupconsisting of STAT3, SOCS3, IFITM2, CEBPD, JUNB, TUBB2A, IL-6ST, CASP4,PROS1, TNFRSF1A, PVRL2, PHF21A, BCL3, NRP1, GLRX, and TGM2 or anortholog or variant thereof.
 2. The composition of claim 1, wherein theprimers include at least five primer pairs selected from the groupconsisting of: TGACATGGAGTTGACCTCG and CTGGAACCACAAAGTTAGTAGTTTC;CCACCTACTGAACCCTCCTCC and TCTTCCGACAGAGATGCTGAA;TCCCACGTACTCTATCTTCCATTC and CTGATGCAGGACTCGGCTG; CGCCATGTACGACGACGAGand CGCCTTGTGATTGCTGTTG; CGACTACAAACTCCTGAAACCG andGAAGAGGCGAGCTTGAGAGAC; AACTTCTCAGATCAATCGTGC and AGACCATGCTTGAGGACAAC;AAGATTTGAAACAGTTGGCATGGAG and CCTTCACTGAGGCATGTAGC;GAGAGACAGCACAATGGGCTC and CTTCCGAAATACTTCCTCTAGGTG;ATCGGATACAGGCCCTAAGTC and TTGTCCAAGACGGCAAGTTG;TGTTACACTAATAGAAACTTGGCAC and CCTTAGGACAGTTCAGCTTGC;AAGCCAAAGAGACTCAGGTG and CAGGTATCAGGGCTGGTTCCTC; GGCAGAAGGAGATGCACAGCand TCAGAGTCTACAGGTTTGGAGAG; CACTCTCTACCAGATAACTGAGGAG andTAATAATTTACATCGTGATCCGTGC; CAACAACTATGATACACCTGAGC andTTCCACTTCACAGCCCAGC; GCAGAGGCTGTGGTCATGC and TGCTTTAATCTTTGCTGGTAGTC;and CTTCACAAGGGCGAACCAC and GCGGCAGACGTACTCCTCAG.


3. The composition of claim 1 or 2, wherein the amplified genes are atleast 90% identical to or specifically hybridize to at least 5 geneshaving accession numbers selected from the group consisting ofNM_(—)213662, NM_(—)003955, NM_(—)006435, NM_(—)005195, NM_(—)002229,NM_(—)001069, NM_(—)002184, NM_(—)001225, NM_(—)000313, NM_(—)001065,NM_(—)002856, NM_(—)016621, NM_(—)005178, NM_(—)003873, NM_(—)002064,and NM_(—)004613.
 4. The composition of claim 1 or 2, wherein theamplified genes are at least 5 genes having accession numbers selectedfrom the group consisting of NM_(—)213662, NM_(—)003955, NM_(—)006435,NM_(—)005195, NM_(—)002229, NM_(—)001069, NM_(—)002184, NM_(—)001225,NM_(—)000313, NM_(—)001065, NM_(—)002856, NM_(—)016621, NM_(—)005178,NM_(—)003873, NM_(—)002064, and NM_(—)004613.
 5. The composition of anyone of claims 1 to 4, which includes primers for amplification of atleast 5, at least 6, at least 7, at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, or at least 15 of saidgenes.
 6. The composition of any one of claims 1 to 4, which includesprimers for amplification of at least 10, at least 11, at least 12, atleast 13, at least 14, or at least 15 of said genes.
 7. The compositionof any one of claims 1 to 4, which includes primers for amplification ofat least 10 of said genes.
 8. The composition of any one of claims 1 to4, which includes primers for amplification of all 16 of said genes. 9.The composition of any one of claims 1 to 4, wherein said primers are incontact with the sample to be tested for the level of IL-6/STAT3 pathwayactivity.
 10. The composition of any one of claims 1 to 9, wherein atleast one of said primers comprises a fluorophore and matchedfluorescence quencher.
 11. The composition of any one of claims 1 to 10,wherein said primers are contained in one or more wells of a multi-wellreaction vessel.
 12. The composition of any one of claims 1 to 11,wherein the primers for amplification of at least two of said genes areincluded together in a duplex or multiplex reaction.
 13. The compositionof any one of claims 1 to 12, further comprising detecting theexpression level of between 1 and 10 housekeeping genes.
 14. Thecomposition of any one of claims 1 to 13, further comprising a DNA orRNA polymerase.
 15. The composition of any one of claims 1 to 14, whichis adapted for effecting PCR, real-time PCR, strand displacementamplification (SDA), loop-mediated isothermal amplification (LAMP),rolling circle amplification (RCA), transcription-mediated amplification(TMA), self-sustained sequence replication (3SR), nucleic acid sequencebased amplification (NASBA), reverse transcriptase polymerase chainreaction (RT-PCR), or helicase-dependent isothermal DNA amplification.16. The composition of any one of claims 1 to 15, wherein said step ofdetecting the level of expression is effected using a double strandnucleic acid-specific dye.
 17. The composition of claim 16, wherein saiddouble strand nucleic acid specific dye is selected from the groupconsisting of SYBR Green I, SYBR Gold, ethidium bromide, propidiumbromide, Pico Green, Hoechst 33258, YO-PRO-I and YO-YO-I, Boxto,Evagreen, LC Green, LC Green Plus, and Syto
 9. 18. The composition ofany one of claims 1 to 17, which is for effecting real-time PCRamplification with detection by a SYBR Green method.
 19. A method forthe detection of IL-6/STATS signaling pathway activity or regulationstatus in a cell sample or subject, comprising use of a compositionaccording to any one of claims 1 to 18 to amplify and detect the levelof expression of at least five genes selected from the group consistingof STATS, SOCS3, IFITM2, CEBPD, JUNB, TUBB2A, IL-6ST, CASP4, PROS1,TNFRSF1A, PVRL2, PHF21A, BCL3, NRP1, GLRX, and TGM2 or an ortholog orvariant thereof.
 20. The method of claim 19, further comprisingcomparing the level of expression of said genes in the sample or subjectto the level of expression of said genes in a control sample.
 21. Themethod of claim 20, wherein said levels of expression are classifiedusing a mathematical classifier method to determine the regulationstatus of IL-6/STAT3 signaling pathway in the in the sample or subjectas compared to the control sample.
 22. The method of claim 21, whereinthis method is a Random Forest method.
 23. The method of any one ofclaims 19 to 22, wherein said step of detecting the level of expressionis effected by a method comprising amplification of mRNA of said atleast two genes.
 24. The method of any one of claims 19 to 23, whereinsaid amplification is effected by a method comprising PCR, real-timePCR, strand displacement amplification (SDA), loop-mediated isothermalamplification (LAMP), rolling circle amplification (RCA),transcription-mediated amplification (TMA), self-sustained sequencereplication (3SR), nucleic acid sequence based amplification (NASBA),reverse transcriptase polymerase chain reaction (RT-PCR), orhelicase-dependent isothermal DNA amplification.
 25. The method of anyone of claims 19 to 24, wherein said step of detecting the level ofexpression is effected using a double strand nucleic acid-specific dye.26. The method of claim 25, wherein said double strand nucleic acidspecific dye is selected from the group consisting of SYBR Green I, SYBRGold, ethidium bromide, propidium bromide, Pico Green, Hoechst 33258,YO-PRO-I and YO-YO-I, Boxto, Evagreen, LC Green, LC Green Plus, and Syto9.
 27. The method of any one of claims 19 to 26, wherein at least one ofsaid primers comprises a fluorophore and matched fluorescence quencher.28. The method of any one of claims 19 to 27, wherein said primers arecontained in one or more wells of a multi-well reaction vessel.
 29. Themethod of any one of claims 19 to 28, wherein the primers foramplification of at least two of said genes are included together in aduplex or multiplex reaction.
 30. The method of any one of claims 19 to29, further comprising detecting the expression level of between 1 and10 housekeeping genes.
 31. A method of determining the level of activityor regulation status of IL-6/STAT3 signaling pathway in a cell sample orin a subject, comprising (a) detecting the expression of at least 2genes selected from the group consisting of STAT3, SOCS3, IFITM2, CEBPD,JUNB, TUBB2A, IL-6ST, CASP4, PROS1, TNFRSF1A, PVRL2, PHF21A, BCL3, NRP1,GLRX, and TGM2, or an ortholog or variant thereof, in a cell sample orsubject; and (b) comparing the expression level of the genes in the cellsample or subject to the expression level of the same genes in a controlcell sample, wherein the level of activity or regulation status of theIL-6/STAT3 signaling pathway in a cell sample or subject is determinedbased on this comparison.
 32. The method of claim 31, wherein at leasttwo primer pairs selected from the group consisting of:TGACATGGAGTTGACCTCG and CTGGAACCACAAAGTTAGTAGTTTC; CCACCTACTGAACCCTCCTCCand TCTTCCGACAGAGATGCTGAA; TCCCACGTACTCTATCTTCCATTC andCTGATGCAGGACTCGGCTG; CGCCATGTACGACGACGAG and CGCCTTGTGATTGCTGTTG;CGACTACAAACTCCTGAAACCG and GAAGAGGCGAGCTTGAGAGAC; AACTTCTCAGATCAATCGTGCand AGACCATGCTTGAGGACAAC; AAGATTTGAAACAGTTGGCATGGAG andCCTTCACTGAGGCATGTAGC; GAGAGACAGCACAATGGGCTC andCTTCCGAAATACTTCCTCTAGGTG; ATCGGATACAGGCCCTAAGTC andTTGTCCAAGACGGCAAGTTG; TGTTACACTAATAGAAACTTGGCAC andCCTTAGGACAGTTCAGCTTGC; AAGCCAAAGAGACTCAGGTG and CAGGTATCAGGGCTGGTTCCTC;GGCAGAAGGAGATGCACAGC and TCAGAGTCTACAGGTTTGGAGAG;CACTCTCTACCAGATAACTGAGGAG and TAATAATTTACATCGTGATCCGTGC;CAACAACTATGATACACCTGAGC and TTCCACTTCACAGCCCAGC; GCAGAGGCTGTGGTCATGC andTGCTTTAATCTTTGCTGGTAGTC; and CTTCACAAGGGCGAACCAC andGCGGCAGACGTACTCCTCAG

are used to detect the expression of the at least 2 genes selected fromthe group consisting of STAT3, SOCS3, IFITM2, CEBPD, JUNB, TUBB2A,IL-6ST, CASP4, PROS1, TNFRSF1A, PVRL2, PHF21A, BCL3, NRP1, GLRX, andTGM2, or an ortholog or variant thereof, in a cell sample or subject.33. The method of claim 31, wherein gene expression is assayed by realtime amplification.
 34. The method of claim 33, wherein the detectionmethod comprises SYBR Green based real-time PCR.
 35. The method of claim31, wherein the gene expression data is analyzed using a ΔΔCt method.36. The method of claim 35, wherein the gene expression data is furtheranalyzed using a Random Forest method.
 37. The method of claim 31,wherein a cell sample is obtained from a patient or non-human animalthat is potentially to be treated with a compound or therapy thatmodulates IL-6/STATS signaling and the method is used to predict whethersaid patient or non-human animal will respond to treatment with saidcompound or therapy.
 38. The method of claim 31, wherein a cell sampleis obtained from a patient or non-human animal that has been treatedwith a compound or therapy that modulates IL-6/STAT3 signaling and themethod is used to assess the efficacy of the treatment protocol.
 39. Themethod of claim 31, which is used to evaluate the regulatory status ofIL-6/STAT3 pathway in a sample.
 40. The method of claim 31, which isused to classify a cell sample as having a deregulated or regulatedIL-6/STAT3 signaling pathway.
 41. The method of claim 31, which is usedto determine whether an agent modulates the IL-6/STAT3 signaling pathwayin sample.
 42. The method of claim 31, which is used to predict theresponse of a subject to an agent that modulates the IL-6/STAT3signaling pathway.
 43. The method of claim 42, which is used to assigntreatment to a subject.
 44. The method of claim 31, which is used toevaluate the pharmacodynamic effects of therapies designed to regulateIL-6/STAT3 pathway signaling.
 45. The method of claim 31, which is usedto evaluate the pharmacodynamic effects of therapies for treatment of adisease associated with IL-6/STAT3 pathway dysregulation.
 46. The methodof claim 31, which is used to evaluate toxicity of an agent a compoundor therapy that modulates IL-6/STAT3 signaling.
 47. The method of claim31, which is used to detect a disease associated with IL-6/STAT3 pathwaydysregulation.
 48. The method of claim 31, which is used to identify adisease associated with IL-6/STAT3 pathway dysregulation.
 49. The methodof claim 31, which is used to diagnose a disease associated withIL-6/STAT3 pathway dysregulation or a subject at risk of developing adisease associated with IL-6/STAT3 pathway dysregulation.
 50. The methodof claim 48 or 49, further comprising treating said patient for saiddisease.
 51. The method of claim 31, which is used to assign treatmentto a subject having a disease associated with IL-6/STAT3 pathwaydysregulation.
 52. The method of claim 31, which is used to predicttreatment outcome for a subject having a disease associated withIL-6/STAT3 pathway dysregulation.
 53. The method of claim 31, which isused to monitor treatment efficacy in a subject having a diseaseassociated with IL-6/STAT3 pathway dysregulation.
 54. The method of anyone of claims 47 to 53, wherein the method is used to assess apre-malignant or cancerous inflammatory condition or other diseaseinvolving aberrant cell proliferation characterized by IL-6/STAT3pathway dysregulation.
 55. The method of any one of claims 47 to 53,which is used to identify a non-cancer inflammatory condition or diseasecharacterized by IL-6/STAT3 pathway dysregulation.
 56. The method ofclaim 54, wherein the method is used to assess a precancerous condition.57. The method of claim 54, wherein the disease is cancer.
 58. Themethod of claim 57, which is used to detect metastases.
 59. The methodof any one of claims 47 to 53, which is used to detect inflammationsites in vivo or ex vivo.
 60. The method of claim 57, wherein the canceris selected from lung, breast, esophageal, head and neck, colonic,gastrointestinal, prostate, multiple myeloma, hepatic, ovarian,neuroblastoma, glioblastoma, melanoma, pancreatic adenocarcinoma, renalcell carcinoma, cholangiocellular carcinoma, and various leukemias andlymphomas.
 61. The method of claim 55, wherein the non-cancerinflammatory condition is selected from the group consisting ofhypoferremia of inflammation, acute-phase response to inflammation andinfection, chronic inflammation, inflammation in cardiovascular,systemic juvenile rheumatoid arthritis, Staphylococcusepidermidis-induced peritoneal inflammation, and pulmonary inflammation.62. The method of claim 61, wherein pulmonary inflammation relateddisorders include adult respiratory distress syndrome, shock lung,chronic pulmonary inflammatory disease, pulmonary sarcoidosis, pulmonaryfibrosis and silicosis.
 63. One or more gene expression data setsobtained using any of the methods recited in claims 19 to
 62. 64. Thegene expression data sets of claim 63, which are derived from the sameindividual.
 65. The gene expression data sets of claim 63, which arederived from different individuals.
 66. The gene expression data sets ofclaim 64 or 65, which are derived from the same or different individualtreated with a particular agent or therapeutic regimen.
 67. A geneexpression data set according to claim 66, wherein the samples areannotated to identify one or more variables such as gender, age, diseasecondition, HLA type, treatment regimen, genetic deficiency.
 68. The geneexpression set of any one of claims 63 to 67, which is suitable for useas part of a therapeutic assessment and/or design of a treatmentregimen.
 69. The gene expression set of any one of claims 63 to 68,which is suitable for use in the design of a therapeutic planningregimen.
 70. A method of using a gene expression set according to anyone of claims 62 to 69 as part of a therapeutic assessment and/or designof a treatment regimen.
 71. The method of any one of claims 19 to 62,which is used in a screen for compounds which modulate Notch signalingpathway activity.
 72. The method of claim 71, comprising: contacting oneor more cells with a compound that potentially modulates Notch pathwayactivity and detecting the level of activity or regulation status of theNotch signaling pathway in said cells, and, based thereon, identifyingsaid compound as a compound that modulates Notch pathway activity. 73.The method of claim 72, wherein said one or more cells are furthercontacted with an agent known to affect Notch pathway activity.